Pharmaceutical compositions comprising lignans and their derivatives for treating hyperplastic diseases

ABSTRACT

The present invention relates to a pharmaceutical composition comprising specific compounds which may be obtained from  Leontopodium alpinum  Cass. (Edelweiss). A preferred compound is leoligin (=(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran-3-yl]methyl(2Z)-2-methylbut-2-enoat]). Corresponding means and methods in respect of medical uses of the compounds are described. The present invention also provides a medical device comprising, containing or having been contacted with the compound. The compounds provided herein may particularly be used in the treatment of hyperplastic diseases, in particular intimal hyperplasia, e.g. stenosis, restenosis, atherosclerosis and the like. Also envisaged herein is the use of these compounds in the treatment of proliferative diseases, such as leukemia, prostate cancer and lung cancer.

The present application is a continuation of U.S. application Ser. No.13/054,385, filed Apr. 19, 2011, which is a national phase applicationunder 35 U.S.C. §371 of International Application No. PCT/EP2009/059256,filed Jul. 17, 2009, which claims benefit of priority to U.S.Provisional Application No. 61/135,287, filed Jul. 18, 2008. The entiretext of each of the above referenced disclosures is specificallyincorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a pharmaceutical composition comprisingspecific compounds which may be obtained from Leontopodium alpinum Cass.(Edelweiss), or structurally related compounds. A preferred compound isleoligin(=(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran-3-yl]methyl(2Z)-2-methylbut-2-enoat]). Corresponding means and methods in respectof medical uses of the compounds are described. The present inventionalso provides a medical device comprising, containing or having beencontacted with the compound. The compounds provided herein mayparticularly be used in the treatment of hyperplastic diseases, inparticular intimal hyperplasia, e.g. stenosis, restenosis,atherosclerosis and the like. Also envisaged herein is the use of thesecompounds in the treatment of proliferative diseases, such as leukaemia,prostate cancer and lung cancer.

Coronary artery bypass grafting (CABG) and percutaneous coronaryintervention (PCI) are the two invasive options to treat coronary arterydisease (CAD), being one of the leading causes of morbidity andmortality worldwide; see WHO, Cardiovascular diseases, InternetCommunication (2007); see also www.who.int/cardiovascular_diseases/en/.The success of both therapeutic approaches is however often limited byrestenosis and graft failure which are considered as hyperplasticdiseases/disorders. With respect to graft patency rates after CABG thevessels of choice are clearly the internal mammary arteries; seeTatoulis, Ann Thorac Surg, 77(1), 93-101 (2004). However due tolimitations in availability saphenous vein grafts are more frequentlyused in CABG than arterial grafts (e.g. in 2004 at the Innsbruck MedicalUniversity 51% of bypass grafts were saphenous veins; see Schachner,European Surgery 39(2), 72-5 (2007). In past years clinicaloptimization, like graft handling (e.g. “no touch techniques”) and lipidlowering therapy has impressively increased the patency rates ofsaphenous vein conduits and is currently approximately at 60% 10 yearsafter CABG (Schachner (2007) loc. cit; Lau, Semin Vasc Med 4(2), 153-9(2004); Tsui, Eur J Vasc Endovasc Surg 23(3), 202-8 (2002). Still, themajor reasons for a loss of patency at earlier time points arethromboses, neointima formation, and intimal hyperplasia, (10-20% lossof patency after the first year), and graft atherosclerosis later afterCABG (Lau (2004), loc. cit.; Hozumi, Heart 76(4), 317-20 (1996); Marin,J Vasc Surg 18(3):407-14 (1993). Thus, graft disease still significantlylimits the durability of venous bypasses.

Generally, the causative factors and the pathophysiological processesthat underlie vein graft disease are not well understood. It is thoughtthat vein graft disease is a result of a variety of events initiated byvascular damage that does occur due to surgical handling, ischemia, andarterialisation (blood pressure, blood flow) of grafts. This initialdamage is though to provoke adaptive repair processes in the vesselwall, like tissue remodelling (positive and negative) and intimalhyperplasia; see Lau (2004), loc. cit., Hozumi (1996), loc. cit., Marin(1993), loc. cit; Lau, Circulation 4, 114(1 Suppl):1435-1440 (2006). Onone hand this response is vital for the adaptation of the graft to thearterial environment, but an excessive response is thought to give raiseto graft disease that ultimately results in graft failure.

Despite a complex array of intra and inter cellular signalling events inthe development of graft disease after CABG and/or PCI the core elementson the histological level are endothelial damage (denudation) and smoothmuscle cell (SMC) proliferation and infiltration of the intima.Pro-inflammatory signalling due to tissue damage and cellular necrosisbut also as an element of adaptive tissue remodelling is another highlyrelevant factor; see Mitra, Immunol Cell Biol 84(2), 115-24 (2006).Although, the excellent concept of using drug eluting stents/matricesinstead of purely mechanical devices will most likely prevail in PCI-and CABG-based prevention of restenosis and graft failure, at the momentthere is a significant lack of functional drugs, screened or designedprecisely for these applications. Currently used drugs are mainlychemotherapeutic agents developed for cancer or immunosuppressivetherapy, which may be too aggressive or unspecific for the treatment ofrestenosis and graft disease, since also endothelial healing—importantfor the prevention of thromboses—is impaired by these drugs.

Lignans are considered as potential candidate molecules which may beused in the treatment of diseases/disorders associated with thecardiovascular system and will be discussed herein below in more detail.However, only a limited number of publications have reported on theimpact of lignans on the cardiovascular system in general, and only afew different lignans have been tested so far. It is of note that atreatment of hyperplastic diseases/disorders with lignans has not beendescribed in the art. The existing data rather suggest that lignans arecardiovascular protective agents with lipid lowering, anti-oxidative,anti-hypertensive, anti-thrombotic, and anti-inflammatory activities.

A large number of lignan-based cancer therapy studies (in vitro and invivo) showed profound cytotoxicity and cell death induction by thesecompounds, see Kim Planta Med, 68(3), 271-4 (2002) and Lin J CellBiochem 84(3), 532-44 (2002). The use of cytotoxic compounds in thetreatment of a hyperplastic disease/disorder, and in particular veingraft disease, is generally considered as detrimental since also healthycells, such as EC cells can be damaged. Hence, the use of cytotoxiclignans known in the art should be avoided in the treatment of thesediseases. Therefore, there is still a demand for compounds which may beused in the treatment of hyperplastic diseases/disorders and which avoidthe disadvantages of compounds known in the art.

SUMMARY OF THE INVENTION

Thus, the technical problem underlying the present invention is theprovision means and methods for the medical interference in hyperplasticdiseases or hyperplastic disorders.

The technical problem is solved by provision of the embodimentscharacterized in the claims.

Accordingly, the present invention relates to a pharmaceuticalcomposition comprising a compound of formula (I)

wherein

-   -   R¹, R² and R³ are independently selected from H, OH, halogen,        alkyl, or alkoxy; and    -   R⁴, R⁵ and R⁶ are independently selected from H, OH, halogen,        alkyl, or alkoxy;    -   R⁷ is selected from —OR⁸, —N(R^(8′))R⁸, —SR⁸, —C(O)R⁸, —OC(O)R⁹,        —C(O)OR⁹,    -   —N(R^(9′))C(O)R⁹, —C(O)N(R^(9′))R⁹ or —S(O)R⁹; wherein R⁸ and R⁹        are independently selected from alkyl or alkenyl and R^(8′) and        R^(9′) are independently selected from H, alkyl or alkenyl; and        wherein any alkyl or alkenyl group comprised in R⁷ may be        unsubstituted or substituted by one or more substituents,        selected from OH, halogen or alkoxy;    -   X is selected from O, S, C(R¹⁰)R¹⁰ and NR¹⁰ wherein R¹⁰ is H,        alkyl or alkenyl;    -   and the dashed lines in the ring structure containing group X        indicate that the respective bond may be a single or a double        bond;        or any pharmaceutically acceptable salt or solvate thereof.

In a preferred embodiment, the compound of formula (I) comprised in thepharmaceutical composition has the stereochemistry indicated in formula(Ia):

wherein R¹ to R⁷ and X are defined as described herein above.

For the above formulae (I) and (Ia), the following embodiments arepreferred in the context of the invention.

Alkyl substituents, as they may be present as R¹ to R⁶, are preferablyC1 to C6 alkyl groups, more strongly preferred are C1 to C3 alkylgroups, and further preferred is methyl.

Halogen substituents include fluoro-, chloro-, bromo- and iodo-atoms,with preference given to chloro and bromo.

As set out above, X is selected from O, S, C(R¹⁰)R¹⁰ and NR¹⁰; whereinR¹⁰, independently for each occurrence, is H, alkyl or alkenyl.Preferred as alkyl group is a C1 to C6 alkyl group, particularlypreferred are methyl and ethyl. Preferred as an alkenyl group is a C2 toC6 alkenyl group.

Preferably, X is O or NR¹⁰, and particularly preferred is O. Preferredgroups R¹⁰ are H and C1 to C6 alkyl, particularly preferred are H andmethyl.

As further explained above, the dashed lines in the ring structurecontaining group X indicates that the respective bond may be a single ora double bond. The ring structure may contain no double bond, one doublebond or two double bonds at the respective position. Preferred are caseswhere no double bond is present, i.e. the ring structure containinggroup X is a saturated ring.

It is generally preferred that at least one of R¹ to R³ represents analkoxy group, and it is more preferred that two or all three of themrepresent an alkoxy group. Among suitable alkoxy groups, generalpreference is given to C1 to C6 alkoxy groups, more strongly preferredare C1 to C3 alkoxy groups and particular preference is given to methoxygroups. If two of R¹ to R³ represent an alkoxy group, it is preferredthat one of them is R².

In the preferred compounds referred to above, wherein one or two of R¹to R³ represent an alkoxy group, it is further preferred that theremaining groups of R¹ to R³ represent H or an alkyl group, preferablyH. Preferred alkyl groups are C1 to C6 alkyl groups, more stronglypreferred are C1 to C3 alkyl groups, and further preferred is methyl.

It is generally preferred that at least one of R⁴ to R⁶ represents analkoxy group, and it is more preferred that two or all three of themrepresent an alkoxy group. Among suitable alkoxy groups, generalpreference is given to C1 to C6 alkoxy groups, more strongly preferredare C1 to C3 alkoxy groups and particular preference is given to methoxygroups. If two of R⁴ to R⁶ represent an alkoxy group, it is preferredthat one of them is R⁵.

In the preferred compounds referred to above, wherein one or two of R⁴to R⁶ represent an alkoxy group, it is further preferred that theremaining groups of R⁴ to R⁶ represent H or an alkyl group, preferablyH. Preferred alkyl groups are C1 to C6 alkyl groups, more stronglypreferred are C1 to C3 alkyl groups, and further preferred is methyl.

Thus, particularly preferred are compounds wherein four, five or all sixof R¹ to R⁶ are alkoxy, and the remaining groups of R¹ to R⁶, if any,are hydrogen. Mention may be made in this respect specifically ofcompounds wherein R¹ is H and R² and R³ are alkoxy, or all of R¹ to R³are alkoxy; and wherein R⁴ is H and R⁵ and R⁶ are alkoxy, or all of R⁴to R⁶ are alkoxy. Among suitable alkoxy groups, general preference isgiven to C1 to C6 alkoxy groups, more strongly preferred are C1 to C3alkoxy groups and particular preference is given to methoxy groups.

R⁷ is preferably —OC(O)R⁹, —C(O)OR⁹, —N(R^(9′))C(O)R⁹, —C(O)N(R^(9′))R⁹or —S(O)R⁹, i.e. an ester, amide or sulfoxy group, with a particularpreference for the ester groups —OC(O)R⁹or

—C(O)OR⁹. Most preferred as R⁷ is a group —OC(O)R⁹.

R⁸ is preferably an alkyl or alkenyl group which is unsubstituted.Preferred alkyl groups have 2 or more, particularly 3 or more carbonatoms. It is further preferred that they have 14 or less, such as 10 orless, particularly 8 or less or 6 or less carbon atoms. Preferredalkenyl groups have 3 or more carbon atoms. It is further preferred thatthey have 14 or less, such as 10 or less, particularly 8 or less or 6 orless carbon atoms. Independent of the number of carbon atoms, it ispreferred that the alkenyl groups have one C—C double bond.

R^(8′) is preferably H or any alkyl group having 10 or less, such as 8or less, preferably 6 or less carbon atoms, such as methyl, ethyl, orpropyl.

R⁹ is preferably an alkyl or alkenyl group which is unsubstituted.Preferred alkyl groups have 2 or more, particularly 3 or more carbonatoms. It is further preferred that they have 14 or less, such as 10 orless, particularly 8 or less or 6 or less carbon atoms. Preferredalkenyl groups have 3 or more carbon atoms. It is further preferred thatthey have 14 or less, such as 10 or less, particularly 8 or less or 6 orless carbon atoms. Independently of the number of carbon atoms, it ispreferable that the alkenyl groups have one C—C double bond.Particularly preferred as R⁹ is a branched alkenyl group as it occurs inleoligin of the formula —C(CH₃)CH—CH₃. In this group, the methylsubstituents at the double bond may be in E- or Z-configuration withrespect to each other, with preference for the Z-configuration.

R^(9′) is preferably H or any alkyl group having 10 or less, such as 8or less, preferably 6 or less carbon atoms, such as methyl, ethyl, orpropyl.

In a strongly preferred embodiment, the present invention concernspharmaceutical compositions comprising compounds of formula (1) or (1a)wherein X is O; wherein, in the case of formula (1), the ring structurecontaining X has no double bonds; wherein four, five or all six of R¹ toR⁶ are alkoxy, and the remaining groups of R¹ to R⁶, if any, arehydrogen; R⁷ is —OC(O)R⁹ or —C(O)OR⁹, particularly —OC(O)R⁹; and R⁹ isan unsubstituted alkenyl group having one double bond and 8 or lesscarbon atoms or an unsubstituted alkyl group having two or more and 8 orless carbon atoms.

While the invention has been described as relating to pharmaceuticalcompositions, it should be understood that the compounds contained inthese pharmaceutical compositions as active agents play an importantrole in the context of the invention. Thus, the claims also encompasspreferred compounds per se, such as the compounds of formula (1) or (1a)defined above, wherein five or all six of R¹ to R⁶ are alkoxy; theremaining group of R¹ to R⁶, if applicable, is hydrogen; and the ringstructure containing X, the group X and R⁷ are as defined above,including preferred embodiments thereof.

A further strongly preferred embodiment relates to a pharmaceuticalcomposition, wherein the compound of formula (I) has the followingstructure:

The chemical structure given herein above is(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran-3-yl]methyl(2Z)-2-methylbut-2-enoat] also commonly known under the trivial name“leoligin”. Leoligin has been shown in the appended examples as aparticularly strong inhibitor of cell proliferation, in particularproliferation of SMCs. It is known that proliferation of SMCs is acentral mechanism specifically involved in a hyperplasticdisease/disorder, in particular intimal hyperplasia. Proliferation ofSMCs is also involved in vein graft disease, which will be describedherein below in more detail.

The present invention solves the above identified technical problemsince, as documented herein below and in the appended examples, it wassurprisingly found that a lignan derived from the roots of Edelweiss(Leontopodium alpinum Cass.), namely leoligin[(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran-3-yl]methyl(2Z)-2-methylbut-2-enoat] and derivatives thereof exhibit a highlybeneficial effect in a medical setting. Leoligin has been shown hereinto be a stronger inhibitor of vascular smooth muscle cell (SMC)proliferation compared to other compounds known in the art, such aslariciresinol (see in particular Example 2 and FIG. 6; formula givenherein below).

Isolated primary human vascular smooth muscle cells represent thecentral cell type in intimal thickening and intimal hyperplasia. Thus,SMC proliferation and migration is a central mechanism underlyinghyperplastic diseases/disorders and inhibitors of this mechanism mayinterfere with the development and/or progression of theses diseases.

Another, even more surprising finding was that not only leoligin butcompounds of formula (I) in general and leoligin derivatives asdescribed herein inhibit vascular smooth muscle cell (SMC)proliferation. Some derivatives of leoligin, such as the 5-methoxy- and5,5′-dimethoxy-derivative, inhibit SMC proliferation at a comparable orat even lower concentration than leoligin, see Example 3 and FIG. 7. Incontrast thereto, lariciresinol (IC₅₀>100 μM) induced a weak inhibitionof SMC when compared to leoligin (IC₅₀=54.5 μM), and its5-methoxy-derivatives, e.g. 5-methoxy-leoligin (IC₅₀=45.9 μM) and5,5′-dimethoxy-derivatives, e.g. 5,5′-dimethoxy-leoligin (IC₅₀=48.6 μM)after 72 h. Without being bound by theory, it is believed that themethoxy-groups may contribute to an increase in lipophilicity of thecompounds to be used in accordance with the present invention, thuspossibly enhancing and/or facilitating their cellular uptake. This maybe one reason why e.g. 5-methoxy-derivatives of leoligin can be used atcomparable or lower concentrations than leoligin.

Structural formulas of exemplary methoxy-derivatives anddimethoxy-derivatives, which also represent preferred compounds in thecontext of the present invention, are given herein below:

A further advantage of the compound comprised in the pharmaceuticalcomposition of the present invention, in particular leoligin andderivatives thereof, is its property not to induce cell death in SMCs,and most importantly also not in endothelial cells (ECs). Endothelialcells form a thin layer of cells, the so called endothelium, that linethe interior surface of blood vessels. The endothelium forms aninterface between the circulating blood and the rest of the vessel wall.Proper endothelial function is essential for blood vessel integrity andloss of its function is a hallmark for vascular diseases.

The property of the compounds to be used in accordance with the presentinvention not to induce cell death is in strong contrast to compoundsknown in the art, where cytotoxic effects were observed, e.g. thelignans honokiol and magnolol described herein below. In contrast, thecompounds provided herein represent lignans which differ from knownlignans by the lack of toxicity and cell death-inducing activity andalso by their cell cycle inhibitory activity. The compounds of thepresent invention are therefore of particular advantage in the treatmentof (a) hyperplastic disease(s)/disorder(s).

In the appended experimental section herein below it is shown that thecompounds of the present invention comprised in a pharmaceuticalcomposition can successfully be used as inhibitor of intimal hyperplasiain a human saphenous vein organ culture model for graft disease. Thecompounds and in particular leoligin, potently inhibited intimalhyperplasia, and even reversed graft disease in pre-damaged vessels. Ina mouse model for venous bypass graft disease leoligin potentlyinhibited intimal hyperplasia in vivo, and had no negative effect on theintegrity of the vascular endothelium. Such an assessment can also becarried out in larger animals/animal models. An exemplary protocol isprovided in the experimental section herein below. In particular, anexemplary protocol using a porcine animal model (i.e.“Landschwein”) toassess the efficacy of the particular compound known under the trivialname “Leoligin” is given in the appended examples. Pigs (such as thewell-known “Landschwein”, a particular pig race) are a preferred animalmodel of (bypass-) intimal hyperplasia and stenosis to be used incontext of the present invention, since the circulatory system of pigsis very similar to that of humans. A person skilled in the art isreadily in the position to adapt this protocol (e.g. to compounds offormula (I), in particular to (a) (di)methoxy-derivative(s) of leoligin(in various concentrations) or to other large animal models) and assessthat compounds of formula (I) as described herein inhibit intimalhyperplasia also in large animals in vivo. It is apparent from the abovethat results obtained in pigs can, to a large extent, be extrapolated tohumans.

The following parameters/effects define independently of each other atreatment success evaluated for example by the above animal models (e.g.porcine model) of compounds of formula (I), in particular Leoligin(and/or its ((di)methoxy-)derivative(s): 1) An intima thickness and/orintima-media thickness of the treatment group below the control group.2) A smaller number of smooth muscle cells in the intima of thetreatment group compared to the control. 3) The presence of a highernumber of p27 and or p21 positive cells in the treatment group comparedto the control. 4) A smaller degree of neointima formation in thetreatment group compared to the control. 5) A reduced presence of tissueremodelling processes in the treatment group compared to the control. 6)A lower number of pro-inflammatory cells in the vessel wall in thetreatment group compared to the control. 7) An intact endothelium. 8) Aphysiological degree of contractility of the vessels. 9) A low degree ofadhesion molecule expression on the endothelial surface. 10) patency ofgrafts and no signs of thrombus formation. 10) Conserved elasticity ofgrafts in the treatment group compared to the control. And 11) Conservedcontractility of grafts in the treatment group compared to the control.

In the prevention of vein graft failure after CABG the major therapeutictargets are neointima formation and intimal hyperplasia (early tointermediate complications) as well as graft atherosclerosis (long termcomplication). Although some progress has been made in past years amajor limit in current strategies is the lack of appropriate compounds,as mentioned above. Most of the applied agents are too aggressive, andoften cause not only SMC apoptosis and inhibition of proliferation, butalso significantly reduce endothelial viability. Since the closure ofthe vascular endothelium after PCI or CABG is very important in vascularhealing and anti-thrombosis, compounds that are not toxic for ECs areinteresting agents for intra- and extravascular drug eluting stents andmatrices. The compounds provided herein and in particular leoligin haveexactly this active profile. Although leoligin inhibits EC proliferation(see FIG. 4), which may reduce local endothelial healing, a wound repairvia the circulation (EC precursors and circulating ECs) is possible (seeFIG. 5). The reason for this in vivo observation may be due to the factthat leoligin is not toxic for ECs, which could facilitate are-colonisation of denuded vessel areas, and consequently reducethromboses. Compounds of formula (I) and, in particular leoligin and((di)methoxy)derivatives thereof may be advantageously used in thetreatment, prevention and amelioration of (a) hyperplysticdisease(s)/disorder(s), in particular intimal hyperplasia and thrombosesin CABG and PCI.

In sum, it has been surprisingly found in the context of the presentinvention that compounds of formula (I) as described herein above, suchas leoligin and ((di)methoxy)derivatives thereof, can successfully beused in a medical setting for the inhibition of cellular proliferation,in particular the proliferation of SMCs. In contrast to compounds knownin the art, the compounds of the present invention are non-toxic and canbe used at low concentrations. Thus, the pharmaceutical composition ofthe present invention comprising these compounds is particularly usefulin the treatment of hyperplastic diseases/disorders, in particularintimal hyperplasia, such as vein graft disease. The compounds may—dueto their EC preserving character—also be applied in drug eluting stents.

Such a beneficial use of these compounds in a medical setting has notbeen described in the art, even though anti-angiogenic activity of somelignans was reported; see Bai, J Biol Chem 278(37), 35501-7 (2003) andBergman Clin Cancer Res 13(3), 1061-7 (2007). However, these lignans arestructurally different from the compounds provided in the presentinvention.

For example, lignans to be used in the prior art are honokiol andmagnolol which are derived from Magnolia species. The respectiveformulas are given herein below:

It is evident from these formulas that neither honokiol nor magnololhave a structural similarity to the compound to be used in context ofthe present invention. Honokiol, a lignan constituent of the plantMagnolia officinalis, was shown to inhibit cell death in ECs but also topotentiate cell death in vivo and in vitro; see Zhang, Eur J Pharmacol,554(1), 1-7 (2007) and Ahn, Mol Cancer Res 4(9), 621-33 (2006). Honokiolwas further shown to caused an arrest in the G1 phase of the cell cyclein SMCs which was associated by an upregulation of p21/WAF1; see Lee(2006), loc. cit. Magnolol, another lignan isolated from Magnoliaofficinalis was shown to induce cell death in SMCs in a capase-dependentmanner and also to inhibit TNFalpha-mediated VCAM-1 expression as wellas to prevent IL-6-induced STAT3 expression in ECs; see Chen, NaunynSchmiedebergs Arch Pharmacol 368(2), 127-33 (2003); Chen, Br J Pharmacol135(1), 37-47 (2002); Chen Br J Pharmacol 148(2), 226-32 (2006). A studyby Razuvaev (J Vasc Surg 46(1), 108-15 (2007)) reported that thecyclolignan picropodophyllin inhibits intimal hyperplasia after ballooninjury in vivo via an interaction with IGF-receptor and ERK signalling.However, a potential toxicity of picropodophyllin on endothelial cellshas not been tested. A few other lignan type compounds, like flax seedlignans have been tested in different model systems of inflammation,cancer, and cardiovascular diseases. Due to a lack of knowledgeconcerning specificities and characteristics of different lignanshowever the mechanisms underlying the interaction of lignans with thecardiovascular system are not well defined.

Without being bound by theory, one mechanism by which the compound ofthe present invention may exert its effect in a medical setting is itsinduction of a cell cycle arrest in the G1-phase, which is associated byan accumulation of the cell cycle inhibitor p27/KIP. The underlyingmechanism of the effect conferred by the compound used and provided inaccordance with the present invention may be its induction of an arrestin the cell cycle. In contrast to honokiol which causes a cell cyclearrest in the G1 phase by an upregulation of p21 (see Lee, FEBS Lett580(22), 5177-84 (2006)), leoligin leads to an increase in p27/KIPprotein levels. Although the result i.e. a G1 phase arrest is similarfor both compounds, the underlying signalling processes involved, andthe mechanism of action may differ. P27/KIP is well known to bind andthereby inactivate the cyclinE/cdk2 complex which phosphorylates pRB.Phosphorylated pRB looses its ability to inhibit transcription factorslike E2F, which upon release serve as transcription factors forproliferation-promoting genes. Although p27/KIP was also reported tocause an arrest at the G2-S transition in a limited number of cell typesvia an interference with cyclinA/cdk2 and cyclinB/cdk2 complexes (seePagano, Mol Cell 14(4), 414-6 (2004)), in the case of SMCs the usuallyobserved interaction of p27/KIP with cyclinE/cdk2 complexes which leadsto an arrest in the G1 phase seems to be at play. The shift in themolecular weight of p27/KIP-1 at 50 μM leoligin from 27 kD to 58 and 85kD respectively, may indicate the binding of p27/KIP-1 to thecyclinE/cdk2 complex, or an oligomerisation of molecules(dimers/trimers). The 23 kD band may represent a cleavage product ofp27/KIP. The signalling pathways via which leoligin leads to theaccumulation and change in molecular weight of p27/KIP-1 protein remainto be elucidated.

Only recently, Edelweiss root extracts have been chemicallycharacterized; see Dobner et al. (2003), loc. cit. Edelweiss rootextracts show a complex pattern of secondary plant metabolites, ofseveral compound classes like coumarins, lignans, sesquiterpenes,polyacetylens, diterpenes, and others; see Schwaiger, Planta Med,70(10), 978-85 (2004). In general, lignans are polyphenolic plantmetabolites derived from phenylalanine, which are synthesized by thecoupling of two phenylpropanoid units by a bond between the β-positionsin the propane side chains. One of these lignans which has been isolatedfrom the roots of Edelweiss is leoligin-IUPAC name[(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran-3-yl]methyl(2Z)-2-methylbut-2-enoat]. Although it is thus known that roots ofEdelweiss (Leontopodium alpinum Cass.), one of the most popular alpineplants, which has been used in folk medicine for the treatment ofdiarrhea, fever, and “abdominal aches” contain lignans, a medical use ofany of these lignans, and in particular leoligin and its((di)methoxy)derivatives, as an inhibitor of cellular proliferation, inparticular the proliferation of SMCs, has neither been described norproposed in the art. Also a medical use of the isolated compounds of thegeneral formula (I) as given herein above, has not been described in theprior art.

As mentioned above, edelweiss and extracts thereof have been used infolk medicine. However, it is of note that only the upper parts (i.e.flowers, leaves and stems) of the Edelweiss plant have been used becausethese contain the bulk of the biomass and have thus been easieravailable. Historical references from the year 1582 mention thatEdelweiss and its relatives are mainly used for the treatment ofdiarrhea and dysentery; see Tabernaemontanus, J. T. (1582): Das AnderBuch von Kreutern. In: Bauhin, H. (ed.) (1731): D. Jacobi TheodoriTabernaemontani neu vollkommen Kraeuter-Buch. Reprint Basel, König,1731. Verlag Kölbl, Grünwald (München) 1993). Further information on thetraditional use of Edelweiss was collected by several diploma theses onthe usage of Alpine plants in folk medicine, performed at the Instituteof Pharmacognosy of the University of Vienna. Interviews of elderinhabitants of alpine regions in Austria and Northern Italy revealed avariety of local knowledge. In Vorarlberg Edelweiss flower heads wereboiled in milk, preparations of which were used for the therapy ofabdominal aches and diarrhea in humans, and particularly also indomestic stock; see Kiene, Volksmedizin in verschiedenen GebietenVorarlberg, Master Thesis at the University of Vienna (1992); Bitschnau,Arzneidrogen der Volksmedizin im Montafon, Master Thesis at theUniversity of Vienna (1991).

Similar information was also obtained for North-Tyrol, East-Tyrol andSouth-Tyrol, where Edelweiss was, furthermore, used to cure tonsillitis,angina and bronchitis, and as an antipyretic to lower fever; seeKnechtl, Volksmedizinisch verwendete Heilpflanzen und Hausmittel imInntal und umgebenden Seitentälern (Tirol), Master Thesis at theUniversity of Vienna (1992); “Wieser, Volksmedizinische Verwendung vonHeilpflanzen und Hausmitteln im Osttiroler Pustertal mit Seitentilem undim Lesachtal”, Master Thesis at the University of Vienna (1995);Pickl-Herck, Volksmedizinische Anwendung im Norden Südtirols. MasterThesis at the University of Vienna (1995). In Polish traditionalmedicine, L. alpinum was used for the therapy of breast cancer by localapplication of a poultice of the aerial plant parts; see Hartwell, J.Nat. Prod. (Lloydia) 31, 71-170 (1968). Knechtl (1992; loc. cit.) alsodescribes that infusions of edelweiss flowers can be used to amelioratestomach-ache. In particular diarrhea in children is to be treated withmilk in which flowers from edelweiss plants has been boiled; see Knechtl(1992; loc. cit.). Wieser (1995; loc. cit.) points out that upper partsof edelweiss plants are used in folk medicine, since edelweiss plantsare selected out of the cut grass of alpine meadows (i.e. the upperparts of edelweiss plants are collected) and dried. This has beenparticularly described for the Villgratental in Kalkstein (1650 m abovesea level). The edelweiss plant is described in Wieser (1995; loc. cit.)as the “chamomile” of the Alps, since it is used in medicine similar tochamomile. According to Wieser (1995; loc. cit.) an edelweiss infusionis used to ameliorate stomach ache, while edelweiss boiled in milk ishelpful in abdominal cramping. Pickl-Herk (1995; loc. cit.) describesthe following medical use of edelweiss flowers: infusion of flowers isbeneficial in ameliorating stomach ache (in particular caused by fouldrinking water), stomach flatulencies, and diarrhea with vomiting.Again, infusions of edelweiss flowers are intended to be administered inparticular to children. “Edelweissmilch” (i.e. 4-5 flowers boiled in 0.51 milk) is used for the following disorders: diarrhea, vertigo,poisoning (leads to vomiting), snake-bites, blood poisoning,indigestion, abdominal cramping, stomach ache, stomach flatulencies, orhangover; see Pickl-Herk (1995; loc. cit.). Also the use of“Edelweissmilch” in veterinary medicine is disclosed in this document,i.e. the treatment of calves suffering from diarrhea and of calves/cowssuffering from stomach flatulencies is described.

Again, the use of Edelweiss in folk medicine as described in the priorart documents above involves only extracts from the upper parts of theplant, but not of the roots. Also a specific compound contained in thisextract to be used in folk medicine has not been described in thiscontext.

As mentioned above and shown in detail in the experimental sectionherein below, the compound to be used in accordance with the presentinvention or the compound as comprised in the pharmaceutical compositionof the present invention may be obtained from plants belonging to thegenus Leontopodium, optionally followed by standard derivatizationreactions. It is particularly preferred that the compounds providedherein may be obtained from Leontopodium alpinum, in particularLeontopodium alpinum Cass., which is commonly known under the trivialname “edelweiss”. According to another nomenclature “edelweiss” may alsobe known under the scientific term “Leontopodium nivale subsp. alpinum(Cass.) Greuter”. However, the terms “Leontopodium alpinum Cass” and“Leontopodium nivale subsp. alpinum (Cass.) Greuter” refer to the sameplant species and merely reflect a regrouping of the species inbotanical nomenclature. Accordingly, the these terms can be usedinterchangeably in context of the present invention and any definitionsand explanations given herein in respect of Leontopodium alpinum Cass.also applies to Leontopodium nivale subsp. alpinum (Cass.) Greuter,mutatis mutandis, and vice versa.

Of course, it is envisaged herein that the compounds to be usedaccording to the present invention may be obtained from otherLeontopodium species, including but not limited to commercial cultivars,such as Leontopodium hybrids. Accordingly the compounds may be obtainedfrom the following, exemplary Leontopodium species and cultivars: L.catipes (DC.) F. Muell., L. gnaphalioides Hieron., L. japonicum var.sandwicense H. Lé., L. linearifolium Britton, L. meredithae (F. Muell.)F. Muell., L. albogriseum Hand.-Mazz., L. aloysiodorum Hort. exHand.-Mazz., L. alpinum Cass., L. alpinum Colm. ex Willk. & Lange, L.alpinum Cass. subsp. nivale (Ten.) Tutin, L. amrheinii Hort. ex Mollers,L. andersonii C. B. Clarke, L. antennarioides Socz., L. arbusculaBeauverd, L. artemisiifolium Beauverd, L. aurantiacum Hand.-Mazz., L.beerianum Beauverd ex Murr, L. blagoveshczenskyi Vorosch., L. bonatiiBeauverd, L. brachyactis Gand., L. caespitosum Beauverd, L. caespitosumDiels, L. calocephalum Beauverd, L. campestre Hand.-Mazz., L. catipes F.Muell., L. chamaejasme Beauverd, L. charkeviczii V. Yu. Barkalov, L.chuii Hand.-Mazz., L. conglobatum Hand.-Mazz., L. coreanum Nakai, L.dedekensi Beauverd, L. delavayanum Hand.-Mazz., L. discolor Beauverd, L.dubium Beauverd, L. evax Beauverd, L. fangingense Ling, L. faurieiHand.-Mazz., L. fedtschenkoanum Beauverd, L. fimbrilligerum J. R.Drumm., L. fischerianum Beauverd, L. foliosum Beauverd, L. forrestianumHand.-Mazz., L. francheti Beauverd, L. futtereri Diels, L. giraldiiDiels, L. gnaphalioides Hieron. ex Sod., L. gracile Hand.-Mazz., L.haastioides Hand.-Mazz., L. hallaisanense Hand.-Mazz., L.haplophylloides Hand.-Mazz., L. hastatum Beavera, L. hayachinense(Takeda) Hara & Kitam., L. helveticum D. Don ex G. Don, L. himalayanumDC., Leontopodium×intermedium Sunderm., L. jacotianum Beauverd, L.jacotianum Beauverd var. haastioides (Hand.-Mazz.) R. C. Srivastava, L.jamesonii Beauverd, L. japonicum Miq., L. japonicum Miq. f. happoenseHid. Takah. ex T. Shimizu, L. javanicum Zoll. & Mor., L. junpeianumKitam., L. kamtschaticum Komarov, L. krasense Derganc, L. kurilenseTakeda, L. leiolepis Nakai, L. leiolepis Nakai var. crinulosum H. S.Pak, L. leiolepis Nakai var. curvicollum H. S. Pak, L. leontopodinumHand.-Mazz., L. leontopodioides Beauverd, L. leontopodium Karst.,Leontopodium×lindavicum Sunderm., L. linearifolium Britton, L.linearifolium Benth. & Hook. f., L. linearifolium Hand.-Mazz., L.longifolium Ling, Leontopodium×macranthum Sunderm., L. maireanumBeauverd ex Hand.-Mazz., L. makianum Kitam., L. mariae Muell., L.melanolepis Ling, L. meredithae F. Muell., L. micranthum Ling, L.microcephalum (Hand.-Mazz.) Ling, L. microphyllum Hayata, L.monocephalum Edgew., L. monoicum Benth. & Hook. f., L. montisganeshii S.Akiyama, L. muscoides Hand.-Mazz., L. nanum Hand.-Mazz., L. nivale(Ten.) Huet ex Hand.-Mazz., L. nivale (Ten.) Huet ex Hand.-Mazz. subsp.alpinum (Cass.) Greuter, L. niveum Hand.-Mazz., L. nobile Beauverd, L.ochroleucum Beauverd, L. ochroleucum Beauverd subsp. campestre (Ledeb.)V. M. Khanminchun, L. ochroleucum Beauverd subsp. campestre(Hand.-Mazz.) Khanm., L. ochroleucum Beauverd subsp. conglobatum(Turcz.) V. M. Khanminchun, L. ochroleucum Beauverd subsp. conglobatum(Hand.-Mazz.) Khanm., L. omeiense Ling, L. palibinianum Beauverd, L.paradoxum J. R. Drumm., L. perniveum Honda, L. pirinicum Hand.-Mazz., L.pulchellum Beauverd, L. pusillum Hand.-Mazz., L. roseum Hand.-Mazz., L.rosmarinoides Hand.-Mazz., L. sachalinense Miyabe & Kudo, L. sandwicenseRock, L. shinanense Kitam., L. sibiricum Cass., L. sinense Hemsl. exForb. & Hemsl., L. smithianum Hand.-Mazz., L. souliei Beauverd, L.spathulatum Kitam., L. stellatum A. P. Khokhr., L. stoechas Hand.-Mazz.,L. stoloniferum Hand.-Mazz., L. stracheyi C. B. Clarke ex Hemsl., L.subulatum Beauverd, L. suffruticosum Y. L. Chen, L. tataricum Kom., L.thomsonianum Beauverd, L. umbellatum Bluff. & Fingerh., L. villosulum A.P. Khokhr., L. villosum Hand.-Mazz., and L. wilsonii Beauverd.

Of course, the compounds provided herein may also be obtained fromcorresponding cell culture, cell suspension culture or a comparable invitro cultivation technique, such as callus culture and the like. Aperson skilled in the art will be aware of corresponding means andmethods for establishing and maintaining corresponding cultures. In apreferred embodiment of the invention, the cell culture is derived fromroots of Leontopodium species described herein above, in particularLeontopodium alpinum (edelweiss). Most preferably, the cell culture isderived from hairy roots.

Based on his general knowledge and the teaching provided herein askilled person is readily in the position to obtain the compounds to beused herein, in particular leoligin, from Leontopodium species.Generally, the person skilled in the art is capable of preparing anextract from plants belonging to the genus Leontopodium by standardtechniques. A preferred method for extracting these compounds from theroots of Leontopodium alpinum is provided in Example 1 herein below. Anartisan will be aware how to adapt this protocol for extracting thecompounds from further Leontopodium species and in particular from rootsof these plants. A skilled person will also be aware of alternativeprotocols to be used in this context. The term extract is well known inthe art and used accordingly herein. For example, this term may refer topreparations of fluid consistence (fluid extracts and tinctures),semisolid consistence (viscous extracts, syrup concentrate) or solidconsistence (dried extracts), which are usually prepared using fresh ordried plant material.

The extract obtained from Leontopodium species is an extract that isreceived by the use of an organic or non-organic solvent. Suitablesolvents are hexane, heptane, petroleum benzene, acetone, chloroform,dichloromethane, ethyl acetate, diethylether, liquid carbon dioxide,ethanol, ternary butyl methyl ether (tBMe) and mixtures of water andalcohol. The extract may be obtained by extracting the plant material,in particular roots, with any of the solvents separately. It is furtherpossible to subsequently extract the obtained extract with a secondsolvent or mixtures of different solvents. An exemplary, non-limitingsolvent to be used in a first extraction step is hexane. However, any ofthe above solvents can be used in such a first extraction step. Thisfirst extraction step may be followed by (a) subsequent second (orfurther) extraction step with at least one of the above exemplarysolvents, e.g. dichloromethane, chloroform or ternary butyl methyl ether(tBMe). Extraction of the compounds disclosed herein (in particularcompounds of formula (I), such as leoligin and/or its(di)methoxy-derivative(s)) in accordance with the present invention isalso illustrated in the appended examples. Preferably, dichloromethaneand methanol are used as extraction solvents. In subsequent extraction,it is preferred that the compounds are first extracted with n-hexane,followed by a subsequent extraction with dichloromethane, chloroform ortBMe. As shown herein, the lignan content (i.e. content of compounds offormula (I), such as leoligin and/or its (di)methoxy-derivative(s)) canbe increased by a second or further extracting steps using the hereindescribed methods, and in particular the above solvents. Also the use ofchromatographic methods, such as Sephadex-LH20-column chromatography andin particular silica gel column chromatograph is advantageous in thiscontext. As also demonstrated in the appended examples, an increase inthe leoligin content from about 0.7% to about 2.2% can be achieved usingSephadex-LH20-column chromatography. It is shown herein that apronounced increase in the leoligin content from about 1.4% to about 10%can be achieved using silica gel chromatograph.

It is envisaged herein that further chromatographic methods to increasethe content of the herein disclosed compounds (in particular compoundsof formula (I), such as leoligin and/or its (di)methoxy-derivative(s))can used in addition or in the alternative to the above describedmethods. Exemplary, non-limiting chromatographic methods to be used inthis context are reversed phase column chromatography or(semi)-preparative HPLC using water/acetonitrile mixtures or comparablesolvent mixtures known in the art. Alternatively, techniques ofliquid-liquid extractions (discontinuous or continuous methods) can beused to increase the content of the herein disclosed compounds (inparticular compounds of formula (I), such as leoligin and its(di)methoxy-derivative(s)). An exemplary liquid-liquid extraction ishigh speed counter current chromatography using a solvent system of twonot mixable solvents.

The preparation of the basic extract of Leontopodium species, inparticular Leontopodium alpinum, may comprise mechanical pulping,sonication, use of mortars and pestles, freeze-thawing cycles, use ofblenders (like Waring-Blenders, Polytron), liquid homogenization andmaceration (see also appended examples), or e.g. Dounce homogenization,Potter-Elvehjem, French Press etc. In the appended examples, amechanical maceration is used. However, the extracts may be obtained bydisrupting the cells and cells from the Leontopodium species by anymechanical/physical or chemical means, like by use of detergents.

Mechanical methods rely on the use of rotating blades to grind anddisperse large amounts of complex tissue, such as plant leaves, flowers,seeds and in particular roots. The Waring blender and the Polytron arecommonly used for this purpose. Unlike the Waring blender, which issimilar to a standard household blender, the Polytron draws tissue intoa long shaft containing rotating blades.

Liquid-based homogenization is the most widely used cell disruptiontechnique for cultured cells. Cells are lyzed by forcing the cell ortissue suspension through a narrow space, thereby shearing the cellmembranes. Three different types of homogenizers are in common use. ADounce homogenizer consists of a round glass pestle that is manuallydriven into a glass tube. A Potter-Elvehjem homogenizer consists of amanually or mechanically driven Teflon pestle shaped to fit a rounded orconical vessel. The number of strokes and the speed at which the strokesare administered influences the effectiveness of Dounce andPotter-Elvehjem homogenization methods. Both homogenizers can beobtained in a variety of sizes to accommodate a range of volumes. AFrench press consists of a piston that is used to apply high pressure toa sample volume of 40 to 250 ml, forcing it through a tiny hole in thepress. Only two passes are required for efficient lysis due to the highpressures used with this process. It is of note that in more industrialapplications also other, larger devices may be employed to prepare theextracts from Leontopodium species.

Sonication is also a physical disruption commonly used to break opencells. The method uses pulsed, high frequency sound waves to agitate andlyse cells and finely diced tissue. To prevent excessive heating,ultrasonic treatment may be applied in multiple short bursts to a sampleimmersed in an ice bath. Sonication is best suited for volumes <100 ml.

The freeze/thaw method is commonly used to lyse bacterial and cells fromhigher organism. The technique involves freezing a cell suspension in adry ice/ethanol bath or freezer and then thawing the material at roomtemperature or 37° C. This method of lysis causes cells to swell andultimately break as ice crystals form during the freezing process andthen contract during thawing. Multiple cycles are necessary forefficient lysis, and the process can be quite lengthy.

Cells, organisms as well as tissue might be treated with various agentsto aid the disruption process. Chemical substances, such as hexane,petroleum benzene, chloroform, dichloromethane, acetone, ethyl acetate,diethyl ether, ethanol and mixtures of water and alcohol or mixtures ofdifferent solvents may be added during or before mechanical disruption.Lysis can also be promoted by suspending cells in a hypotonic buffer,which cause them to swell and burst more readily under physicalshearing. Processing can be expedited by treating cells with glass beadsin order to facilitate the crushing of cell walls. Viscosity of a sampletypically increases during lysis due to the release of nucleic acidmaterial. DNase may be added to samples along with to reduce thisproblem.

Less preferred, however envisaged, is the use of detergents in thepreparation of the extracts to be treated in accordance with the presentinvention. Detergents are a class of molecules whose unique propertiesenable manipulation (disruption or formation) of hydrophobic-hydrophilicinteractions among molecules in biological samples. Such detergents maybe used to lyse cells, solubilize membrane proteins and lipids.Generally, moderate concentrations of mild (i.e., nonionic) detergentscompromise the integrity of cell membranes, thereby facilitating lysisof cells and extraction of soluble protein, often in native form. Usingother conditions, detergents effectively penetrate between the membranebilayers at concentrations sufficient to form mixed micelles withisolated phospholipids. Detergents may be, e.g. Triton X-100®,Triton-X-114®, NP-40®; CHAPS, Tween-20®, Tween-40®, Tween-80®, OctylGlucoside, Octylthio Glucoside, Brij-35, Brij-58, SDS and the like.However, it may be useful to stabilize the extract by certain chemicalmeans. Illustrative stabilizers are discussed herein below in context ofpharmaceutical or cosmetic compositions.

The cells and plants to be employed in order to obtain the basic extractmay be cells of natural origin as well as cultured cells or plants. Itis preferred herein that the cells or plants and in particular roots ofthe plants are dried before mechanical disruption/maceration asdescribed herein above. The cells or plants may be air dried,lyophilized (freeze-dried) or, though less preferred, dried in an oven.It is preferred herein that the “cell(s)” and “plant(s)” to be used as abasic material are fresh, i.e. harvested shortly before the extract isprepared. Nonetheless, it is possible to store the basic material beforeits use in the preparation of the extract. For example, the basicmaterial may be lyophilized (freeze-dried) or simply frozen and storedat low temperatures, e.g. at about −20 to −30° C. or as low as −80° C.

In context of the present invention, the term “cell” and “plant” to beused as basic material for preparing the extract to be treated by themethod of the present invention also comprises the use of “tissues”.Such tissues may be leaves, sprouts, or reproductive organs e.g.flowers. Preferably, the tissues are roots, in particular hairy roots.In addition, callus or cell cultures may be used which may be derivedfrom tissues described above, in particular roots, and which are grownin liquid culture or on solidified culture medium. The appropriateculturing methods of calli or cell cultures are known to a personskilled in the art. A culture medium may be for example a MS (Murashigeand Skoog) medium while a solidifying agent may be agarose, plant agaror bacto agar. A basic culture medium such as a MS medium may bemodified in respect to pH range, carbon or nitrogen source, amino acidsor vitamins amongst others. The use of plants regenerated from suchcallus or cell culture is also envisaged, as well as plants or organismsgenerally grown or propagated in vitro.

Methods for preparing the extract are known in the art and alsodescribed herein. Preferably, the extract is further processed shortlyafter its preparation (e.g. the extract is used in the preparation of aherein disclosed pharmaceutical composition); however, it is alsopossible to store the extract for some time before they are used inaccordance with the present invention. The extracts may, for example, bestored in lyophilized form or in form of dried extracts. However, eachstorage form known in the art is be employed, as long as the storage hasthe effect that the extract (and its components) remain efficacious overa long time period, i.e. the stored extract has, preferably,substantially the same efficacy as the fresh extract.

Dried extracts can be routinely prepared by methods known in the art.For example, following mechanical disruption of the basic (plant)material by e.g. maceration or percolation, the material can beextracted using (a) solvent(s) or mixtures thereof as described herein.After separation of the fluid phase and the extract residue (whichcontains e.g. cellulose, pectin and the like and which does, preferably,not contain the active substance(s) as disclosed herein, i.e.predominantly leoligin and its ((di)methoxy)-derivative(s). The fluidextract (i.e. the fluid phase of the obtained extract) may beconcentrated taking advantage of routine techniques, some of which areexemplarily described herein below. Such concentration techniquesinclude, but are not limited to fluidised-bed drying, concentration to asyrup or concentrated fluid extract, spray drying, freeze drying or theuse of a vacuum dryer, a drying tunnel, vacuum band dryer or a dryinghurdle. Often organic-hydrous fluid extracts (such as the fluid extractobtained herein using an organic solvent) are concentrated by nucleateboiling or surface evaporation.

Routine drying techniques employed in the pharmaceutical field comprisedistillation and drying under normal conditions (i.e. room temperature)also methods which take advantage of variations in pressure andtemperature in order to obtain the dried extracts. One well known methodfor preparing a dried extract is as follows: First, a fluid extract ortincture is prepared; after subsequent distillation of the solvent aviscous extract is obtained, to which often adjuvants and/or excipients(e.g. lactose, polyvinylpyrrolidone, sucrose, silicon dioxide and thelike are added. This moist mass is then dried in suitable driers. Alsoemployed in this context is the use of a vacuum band dryer (MitchellDryers Ltd), wherein a dried extract is obtained from the viscousextract after a pre-drying step using downdraft vaporizers.

Also envisaged herein is the use of commercially available extracts, inparticular dried extracts, obtained from (a) plant(s) belonging to thegenus Leontopodium.

After mechanical disruption of the cell(s), tissue(s) or whole plant(s)the plant material may be further macerated and/or dissolved/suspendedin an organic solvent, such as hexane, petroleum benzene, chloroform,dichloromethane, acetone, ethyl acetate, diethyl ether, liquid carbondioxide, ethanol and mixtures of water and alcohol with any of thesolvents separately or subsequently with a second solvent or mixtures ofdifferent solvents. Preferably, dichloromethane and methanol are used asextraction solvents.

As shown in the appended examples, a hexane extract comprising 0.67%leoligin and 1.47% leoligin and its methoxy-derivative(s) can easily beprepared by routine techniques. However, it is preferred herein that theextract is enriched in the compounds described and provided herein, inparticular compounds of formula (I), such as leoligin and/or its((di)methoxy) derivative(s). As also shown in the appended examples,higher yields (relative to the leoligin content [w/w %] in the extract)typically in a range between 0.7% to 1.5% can easily be obtained usingstandard extraction methods and solvents (such as dichloromethane).Using these standard extraction methods, yields of up to about 2.2% ofleoligin and its 5-methoxy-derivative can be obtained. As describedherein, the content of compounds of formula (I), in particular leoligin(and/or its (di)methoxy-derivative(s)), can be further increased bymultiple extraction rounds, e.g. a first extraction step using hexanefollowed by (a) subsequent extraction step(s) using e.g.dichloromethane, chloroform or ternary butyl methyl ether (=tBMe). Atotal lignan content (predominantly compounds of formula (I), inparticular leoligin and/or its (di)methoxy-derivative(s)) of at least2.4% can be achieved if subsequent extraction steps are applied Theconcentration of lignans (predominantly compounds of formula (I), inparticular leoligin and/or its (di)methoxy-derivative(s)) can also beincreased by the use of Sephadex-LH20-column chromatography (increase inthe leoligin content from about 0.7% to about 2.2%).

Preferably, the extract is an enriched extract, i.e. contains leoliginand its ((di)methoxy)-derivative(s) in a high amount. Such an enrichedextract can, for example, be obtained by taking advantage of silica gelchromatography as demonstrated in the appended examples. Silica gelcolumn chromatography is well known in the art and described in detailin standard textbooks, such as “Preparative Chromatography Techniques”by Hostettmann, K. Marston, Andrew Hostettmann, Maryse, Springer-VerlagGmbH, 2007, 260 p. In the experimental section, it was shown that apronounced increase in the leoligin content from 1.36% to 9.76% [w/w]can be achieved using silica gel chromatograph (mobile phase: petroleumether-acetone).

Accordingly, it is preferred herein that the solid components of theextract (e.g. after evaporating the solvent by any of the drying methodsdescribed herein) comprise at least 0.05%, 0.1%, 0.5%, 0.7%, 1%, 1.5%,2.0%, 2.5% or 3.0% of the compounds of formula (I), in particularleoligin and/or ((di)methoxy)-derivative(s) thereof, wherein an extractthe solid components of which comprise at least 0.7% of these compoundscan be considered an “enriched” extract in context of the presentinvention. More preferably, the solid components of the extract compriseat least 5%, 6%, 7%, 8%, and most preferably at least 9% or 10% of thecompounds of formula (I), in particular leoligin and/or((di)methoxy)-derivative(s) thereof. An extract, the solid components ofwhich comprise at least 9% of these compounds can be considered a“highly enriched” extract. An “enriched extract”, and, in particular a“highly enriched” extract as defined herein, represents therefore apreferred embodiment of the present. “Enriched” or “highly enriched”extracts are particularly useful in the herein disclosed medicalcontext, in particular the treatment, prevention, or amelioration of (a)hyperplastic disorder(s) as defined herein. In accordance with thepresent invention, it is also preferred herein that the solid componentsof the (highly enriched) extract comprise at least 15%, 20%, 25%, 30%,40%, 50%, 60%, 80% or 90% of the compounds of formula (I), in particularleoligin and/or (a) derivative(s) thereof (preferably (a)(di)methoxy-derivative(s), more preferably the herein disclosedderivatives 5-Methoxy-Leoligin and/or 5,5′-Dimethoxy-Leoligin). Based onthe teaching provided herein a skilled person is readily in the positionto determine whether an extract prepared in accordance with the presentinvention is enriched/highly enriched in compounds of formula (I), inparticular leoligin and/or (a) derivative(s) (preferably (a)(di)methoxy-derivative(s), more preferably the herein disclosedderivatives 5-Methoxy-Leoligin and/or 5,5′-Dimethoxy-Leoligin). Mostpreferably, pure compounds of formula (I) are obtained, i.e. solidcomponents of the extract comprise at least 95% of the compoundsdescribed and provided herein. In order to obtain a higher yield of thecompounds of formula (I), the basic extracted material may be subjectedto at least one further and up to eight further cycles of extraction. Itis preferred that the (enriched/highly enriched) extract is obtainedfrom (a) plant(s) belonging to the genus Leontopodium, in particularfrom the roots of such (a) plant(s). Exemplary species or cultivars ofthe above genus and to be used in accordance with the present inventionare known in the art and also disclosed herein.

It is envisaged herein, that the “enriched/highly enriched” extractcomprises predominantly leoligin as active substance, in particular incombination with its methoxy-derivatives. Based on the teaching givenherein, a skilled person is readily in the position to determine whichamount of the (enriched/highly enriched) extract is to be employed inparticular in the preparation of the pharmaceutical compositionscomprising/consisting of the extract depending on theconcentration/content of the herein disclosed active substance(preferably of leoligin and/or its (di)methoxy-derivative(s) andmixtures thereof). Preferably, the extract employed/contained in thepharmaceutical composition exerts substantially the same medical effectas a pharmaceutical composition comprising (a) compound(s) of formula(I), in particular leoligin and/or its (di)methoxy-derivative(s) (ormixtures thereof), more particularly leoligin or the(di)methoxy-derivative alone (as shown in the appended examples).“Substantially the same effect” means in context of the presentinvention that the “effect” varies by less than 10%, preferably lessthan 5%, most preferably less than 1%. An exemplary “effect” to bemeasured is inhibition of vascular smooth muscle cell (SMC)proliferation which is a central mechanism underlying hyperplasticdiseases/disorders as described herein above and also demonstrated inthe appended examples.

As mentioned above, the herein provided and disclosed extracts obtainedfrom (a) plant(s) belonging to the genus Leontopodium can, in accordancewith the present invention, be used in a medical context. Accordingly,the present invention relates in one embodiment to a pharmaceuticalcomposition comprising a root extract obtained from a plant belonging tothe genus Leontopodium. A further embodiment relates to a pharmaceuticalcomposition comprising an extract obtained from a plant belonging to thegenus Leontopodium, whereby the extract is highly in enriched in theherein disclosed compounds, in particular compounds of formula (I), suchas leoligin and/or its (di)methoxy-derivative(s) (or mixtures thereof).It is preferred herein that the highly enriched extract predominantlycomprises leoligin and/or its (di)methoxy-derivative(s) (or mixturesthereof). Formulas of leoligin and preferred (di)methoxy-derivativesthereof are also provided herein. The herein disclosed pharmaceuticalcomposition comprising a (root) extract obtained from a plant belongingto the genus Leontopodium, wherein the extract is preferably enriched(most preferably highly enriched) in the compounds of formula (I), inparticular leoligin and/or ((di)(methoxy-))derivatives thereof, is usedin the treatment, prevention or amelioration of a hyperplastic disorderas defined herein. The term “root extract” used herein means an extractobtained from roots, i.e. plant material from the lower parts of theplants are used, preferably only roots are used as raw plant material inthe preparation of the extract. It is preferred in this context that thepharmaceutical composition consists of the (preferably enriched, morepreferably highly enriched) extract. However, furtherexcipients/adjuvants/carriers and the like as described herein and knownin the art may be contained in the pharmaceutical composition inaddition to the extract. In accordance with the above, a compositioncomprising (consisting of) a(n) (root) extract obtained from a plantbelonging to the genus Leontopodium, whereby the extract is (highly) inenriched in compounds of formula (I), in particular leoligin and/or its(di)methoxy-derivative(s) (or mixtures thereof), is provided herein foruse in medicine or for use as a medicament. Also a (root) extractobtained from a plant belonging to the genus Leontopodium, whereby theextract is (highly) in enriched in compounds of formula (I), inparticular leoligin and/or its (di)methoxy-derivative(s) (or mixturesthereof), for use in medicine or as a medicament is provided. It isenvisaged that the above (pharmaceutical) compositions/extracts are tobe used in accordance with the present invention in the treatment,prevention or amelioration of a hyperplastic disorder as disclosed anddefined herein.

In order to obtain the single compounds, the extracts may be preparedand evaporated as described above and, submitted to further purificationby column chromatography using silica gel, silica gel modified by meansof AgNO₃, reversed phase material (RP18) or Sephadex LH 20® asstationary phases. Additionally, other separation techniques e.g. highspeed counter current chromatography or (semi)-preparative HPLC might beused as well. Fractions obtained by the above mentioned chromatographictechniques may be further purified, e.g. by another cycle ofchromatographic purification. For example, a cross-linked dextran gelmay be used for such further purification, like e.g. Sephadex LH-20®.This kind of chromatography is usually performed in the presence of anorganic solvent such as methanol, acetone dichloromethane and the like.It is envisaged herein that the herein described pharmaceuticalcompositions comprising the extract disclosed herein may also (inaddition) comprise the pure (and/or (substantially) purified, e.g.purified from the extract) active substances (i.e. compound of formula(I), in particular Leoligin and/or its (di)methoxy derivative(s)). Inaccordance with the above, it is preferred herein that thepharmaceutical composition comprises essentially the plant extractsdisclosed herein and obtained by the herein described methods. Alsoenvisaged herein is a pharmaceutical composition, which does notcomprise the herein described extract, but comprises the pure (and/or(substantially) purified, e.g. purified from the extract) activesubstances (i.e. compound(s) of formula (I), in particular Leoliginand/or its (di)methoxy-derivative(s)). The extract can also be obtainedby alternative extraction methods known in the art e.g. supercriticalcarbon dioxide extraction, percolation or Soxhlet-extraction andadaptable for the means and methods of the present invention by oneskilled in the art.

Also envisaged herein, though less preferred, the compounds may also beobtained from upper parts of the plants, e.g. flowers, stems, leaves,seeds and the like. Leontopodium alpinum (Edelweiss) plants to beextracted are easily available e.g. from the Station féderale derecherches en production végetale de Changins (see alsohttp://www.admin.ch/sar/rac; Revue Suisse Vitic. Arboric. Hortic. 31(2),889-96 (1999)).

In an alternative embodiment, the compounds to be used herein may alsobe synthesized. An exemplary synthetic pathway of leoligin is shown inFIG. 8. The shown synthetic pathway might be adapted by a change of thecorresponding educts to obtain other compounds of the present inventionA skilled person will be aware of methods of synthesizing the compoundsof the present invention, in particular leoligin, or may deducecorresponding methods e.g. from Li Hong Hu, J. Nat. Prod. 68, 342-8.(2005); Babasaheb P. Bandgar, Monatshefte für Chemie 135, 1251-5 (2004);J Pijus Kumar Mandal, Org. Chem. 63, 2829-34 (1998); Subhas Chandra Roy,J. Org. Chem. 67, 3242-8 (2002).

As mentioned above, the active compounds referred to herein may also beprovided via semi-synthetic methods, e.g. by derivatizing a naturalproduct such as leoligin. Suitable derivatization reactions known in theart comprise methods wherein the ester bond present in leoligin issaponified to produce an alcohol. The alcohol may be oxidized to providea carbonyl/carboxylic acid functionality to be reacted with an alcohol,thiol or amine, or it may be esterified with a different organic acid,it may be converted into an amine etc.

The pharmaceutical composition may comprise the compounds provided inthe present invention. The compounds to be used in accordance with thepresent invention may be obtained from Leontopodium plants as describedherein above and/or chemically synthesized.

The pharmaceutical composition of the present invention comprisingcompounds of formula (I) and, in particular, leoligin, will beformulated and dosed in a fashion consistent with good medical practice,taking into account the clinical condition of the individual patient,the site of delivery of the pharmaceutical composition, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” of the pharmaceuticalcomposition for purposes herein is thus determined by suchconsiderations.

The skilled person knows that the effective amount of pharmaceuticalcomposition administered to an individual will, inter alia, depend onthe nature of the compound. For example, if said compound is a lignanthe total pharmaceutically effective amount of pharmaceuticalcomposition administered parenterally per dose will be in the range ofabout 1 μg/kg/day to 10 mg/kg/day of patient body weight, although, asnoted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day. If given continuously,the pharmaceutical composition is typically administered at a dose rateof about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injectionsper day or by continuous subcutaneous infusions, for example, using amini-pump. An intravenous bag solution may also be employed. The lengthof treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect. The particular amounts may be determined by conventionaltests which are well known to the person skilled in the art.

Pharmaceutical compositions of the invention may be administered orally,rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, drops ortransdermal patch), bucally, or as an oral or nasal spray. Accordingly,also the compound provided herein may be administered by any one of aparenteral route, oral route, intravenous route, intraarterial route,intramuscular route, intracardial route, intrapulmonal route,intravesical route, intravitreal route, subcutaneous route, intranasalroute or transdermal route.

Pharmaceutical compositions of the invention preferably comprise apharmaceutically acceptable carrier. By “pharmaceutically acceptablecarrier” is meant a non-toxic solid, semisolid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion.

The pharmaceutical composition is also suitably administered bysustained release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or microcapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. etal., Biopolymers 22:547-556 (1983)), poly(2-hydroxyethyl methacrylate)(R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R.Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langeret al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustainedrelease pharmaceutical composition also include liposomally entrappedcompound. Liposomes containing the pharmaceutical composition areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046;EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes areof the small (about 200-800 Angstroms) unilamellar type in which thelipid content is greater than about 30 mol. percent cholesterol, theselected proportion being adjusted for the optimal therapy.

For parenteral administration, the pharmaceutical composition isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation.

Generally, the formulations are prepared by contacting the components ofthe pharmaceutical composition uniformly and intimately with liquidcarriers or finely divided solid carriers or both. Then, if necessary,the product is shaped into the desired formulation. Preferably thecarrier is a parenteral carrier, more preferably a solution that isisotonic with the blood of the recipient. Examples of such carriervehicles include water, saline, Ringer's solution, and dextrosesolution. Non aqueous vehicles such as fixed oils and ethyl oleate arealso useful herein, as well as liposomes. The carrier suitably containsminor amounts of additives such as substances that enhance isotonicityand chemical stability. Such materials are non-toxic to recipients atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, succinate, acetic acid, and other organic acids ortheir salts; antioxidants such as ascorbic acid; low molecular weight(less than about ten residues) (poly)peptides, e.g., polyarginine ortripeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids, such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, manose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;counterions such as sodium; and/or nonionic surfactants such aspolysorbates, poloxamers, or PEG.

The components of the pharmaceutical composition to be used fortherapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes (e.g.,0.2 micron membranes). Therapeutic components of the pharmaceuticalcomposition generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The components of the pharmaceutical composition ordinarily will bestored in unit or multi-dose containers, for example, sealed ampoules orvials, as an aqueous solution or as a lyophilized formulation forreconstitution. As an example of a lyophilized formulation, 10-ml vialsare filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution, andthe resulting mixture is lyophilized. The infusion solution is preparedby reconstituting the lyophilized compound(s) using bacteriostaticWater-for-Injection.

In one embodiment, a compound as defined herein above is for use intreating, preventing or ameliorating of a hyperplastic disease/disorder.The present invention also relates to the use of a compound as definedherein for the preparation of a pharmaceutical composition for thetreatment, prevention or amelioration of a hyperplasticdisease/disorder. Hyperplasia/Hyperplastic diseases/disorders (malignand benign) are characterised by abnormal (i.e. hyperphysiological) highnumbers of cell divisions in an organ or tissue. Hyperplasia may be apathological or a physiological process which is however always clearlydistinguishable from hypertrophy. Hypertrophy is the enlargement oftissue or an organ by enlargement of single cells. Hyperplasia is thephysiologic or pathological enlargement of tissues or organs due to anincrease in the number of cells due to cell division. An important partof vein graft disease is a process called intimal hyperplasia, being ahyperplastic process, which leads to narrowing of the vessel lumen andgraft failure. Another process that contributes to vein graft disease isatherosclerosis, which is characterised by deposition of lipids in thevessel wall, infiltration of macrophages, foam cell formation and fattystreak formation, tissue remodelling which is associated with theproliferation and infiltration of the intima by smooth muscle cells,deposition of extracellular matrix, and plaque formation. In addition,inflammatory processes crucially contribute to atherosclerosis, veingraft disease and hyperplasia. These processes may lead to plaquerupture which may lead to the thrombosis and or occlusion of the veingraft. In the process of atherosclerosis proliferation of smooth musclecells plays an important role, also this proliferation is a hyperplasticprocess.

A preferred hyperplastic disease to be treated, prevented or amelioratedin accordance with the present invention is intimal hyperplasia and/orvein graft disease. Vein graft disease is characterised by intimalhyperplasia i.e. the abnormally increased proliferation of smooth musclecells in the intima and media of the vessel wall. Preferably, thehyperplastic disease/disorder is hyperplasia. The hyperplasia may beintimal hyperplasia. In a preferred embodiment the intimal hyperplasiais stenosis or restenosis. The intimal hyperplasia may also beatherosclerosis. The meaning of the terms “hyperplasia”, “intimalhyperplasia”, “stenosis”, “restenosis” and “atherosclerosis” is wellknown in the art and may be deduced from standard textbooks such as“Handbook of Coronary Stents” edited by P. W. Serruys and B. Rensing,4th edition, published by Taylor&Francis, or from the “Handbook ofDrug-eluting stents, edited by P. W. Serruys and A. H. Gershlick,published by Informa Healthcare. It is particularly envisaged hereinthat vein graft diseases may be treated in accordance with the presentinvention.

The hyperplastic disease may also be a proliferative or neoplasticdisease. A proliferative disease is generally considered as a diseaseassociated with uncontrolled/increased proliferation of cells. Neoplasiaor neoplastic diseases are charaterised by the new formation of tissueseither as a physiological process or as a pathological process. Typicalpathological neoplastic diseases are tumors/cancers. Non-limitingexamples of proliferative diseases are benign proliferative diseases,such as benign proliferative breast disease, cancerous disorders, likeblood tumors, leukemia, as well as solid tumors like B-cell lymphomas,myelotic cancer, prostate cancer, breast cancer, colon cancer, lungcancer and skin cancer.

In one embodiment, the present invention relates to a method fortreating, preventing or ameliorating a hyperplastic disease/disordercomprising the administration of a compound as defined herein to asubject in need of such a treatment, prevention or amelioration. It ispreferred that the subject is a human.

A preferred application form is a drug eluting stent system describedherein may be a polymer based drug delivering system or a polymer coateddrug delivering system. It is to be understood that the compound offormula (I) described and provided herein, and in particular leoliginand/or (a) ((di)methoxy)-derivative(s) thereof, is applied to the drugdelivering system in combination with (a) polymer(s). Therefore the drugcomponent (the active ingredient) is embedded in a non-erodible polymercarrier (base coat formulation) which is surrounded by a suitabletopcoat layer to control the release of the embedded drug. A possibleapplication form would be a system containing parylene C and thefollowing two non-erodible polymers: polyethylene-co-vinyl acetate(PEVA) and poly n-butyl methacrylate (PBMA). A combination of the twopolymers (67%/33%) mixed with the compound of formula (I) described andprovided herein, and in particular leoligin and/or (a)((di)methoxy)-derivative(s) thereof, makes up the basecoat formulationwhich is applied to a parylene C treated stent. A drug-free topcoat ofPBMA polymer is applied to the stent surface to control the releasekinetics of the compound of formula (I) described and provided herein,and in particular leoligin and/or (a) ((di)methoxy)-derivative(s)thereof. Alternatively a single layer polymer e.g. a Translute® polymercarrier, might be used as drug delivering matrix. The drug/polymercoating is adhered to the entire surface (i.e., luminal and abluminal)of the stent.

In one embodiment, the present invention relates to a medical devicecomprising, containing or having been contacted with a compound asdescribed herein, i.e. a compound with formula (I), in particularleoligin.

Preferably, the medical device is a drug delivering system. Such a drugdelivering system may, for example, be a balloon catheter. A ballooncatheter is a kind of a catheter having an inflatable balloon at itstip. The balloon catheter can be used to widen a narrow opening orpassage within the human or animal body, and may be of particularadvantage in the treatment of a hyperplastic disease like e.g. intimalhyperplasia, restenosis, stenosis or vein graft disease. In a firststep, the deflated balloon catheter is positioned at the side to bewidened and is then in a second step inflated. After widening theopening or passage the balloon is deflated and can then easily beremoved. It is envisaged that a balloon used in this context may becoated or may contain the compound of formula (I), in particularleoligin and/or its ((di)methoxy)-derivative(s). These compounds may bedelivered to the cells or tissue surrounding the balloon during and/orupon insertion, inflation and deflation of the balloon. A balloon asdescribed herein above may be particularly beneficial in this contextsince it can be completely removed after the widening procedure,reducing the possible occurrence of inflammatory responses.

The drug delivering system described herein may be a polymer based drugdelivering system or a polymer coated drug delivering system. It is tobe understood that the compound of formula (I) described and providedherein, and in particular leoligin and/or ((di)methoxy)-derivative(s)thereof, is applied to the drug delivering system in combination with(a) polymer(s). Preferably, the drug delivering system provides for asustained release of the compounds. Non-limiting examples of polymer tobe used for coating the drug delivering systems are polyethylene glycol;polystyrene; polyurethane; poly(hydroxyvalerate); poly(L-lactic acid);polycaprolactone; poly(lactide-co-glycolide); poly(hydroxybutyrate);poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoester;polyanhydride; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolicacid-co-trimethylene carbonate); polyphosphoester; polyphosphoesterurethane; poly(amino acids); cyanoacrylates; poly(trimethylenecarbonate); poly(iminocarbonate); copoly(ether-esters) (e.g., PEO/PLA);polyalkylene oxalates; polyphosphazenes; biomolecules, such as fibrin,fibrinogen, cellulose, starch, collagen and hyaluronic acid;polyurethanes; silicones; polyesters; polyolefins; polyisobutylene andethylene-alphaolefin copolymers; acrylic polymers and copolymers; vinylhalide polymers and copolymers, such as polyvinyl chloride; polyvinylethers, such as polyvinyl methyl ether; polyvinylidene halides, such aspolyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile;polyvinyl ketones; polyvinyl aromatics, such as polystyrene; polyvinylesters, such as polyvinyl acetate; copolymers of vinyl monomers witheach other and olefins, such as ethylene-methyl methacrylate copolymers,acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetatecopolymers; polyamides, such as Nylon 66 and polycaprolactam; alkydresins; polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxyresins; polyurethanes; rayon; rayon-triacetate; cellulose; celluloseacetate; cellulose butyrate; cellulose acetate butyrate; cellophane;cellulose nitrate; cellulose propionate; cellulose ethers; andcarboxymethyl cellulose.

Preferably, the medical device provided herein is a stent. In context ofthe present invention the term “stent” means a medical device that isinserted into a natural conduit of the body to prevent or counteract adisease-induced localized flow constriction. Stents provided herein maypreferably be used in the treatment of (a) hyperplasticdisease(s)/disorder(s), in particular (a) vein graft disease(s). Themeaning of the term “drug eluting stent” is well known in the art andmay, for example, be deduced from the “Handbook of Coronary Stents”edited by P. W. Serruys and B. Rensing, 4^(th) edition, published byTaylor&Francis, or from the “Handbook of Drug-eluting stents, edited byP. W. Serruys and A. H. Gershlick, published by Informa Healthcare.Based on his general knowledge and the teaching provided herein and instandard text books, like the above-mentioned Handbook of CoronaryStents, a person skilled in the art is readily in the position todevelop and prepare a stent and in particular a drug eluting stentcomprising, containing or having been contacted with a compound offormula (I), in particular leoligin and/or its((di)methoxy)-derivative(s). A skilled person will be aware of methodsfor coating the stent with the herein described and provided compound,whereby the coating allows delivery of the compound to cells and/ortissue, e.g endothelial cells/endothelium. The stent, in particular thedrug eluting stent, to be used herein may also be a biodegradable stent,i.e. the stent is degraded/dissolved some time after insertion of thestent in a passage to be widened as described herein above.

In a preferred embodiment of the present invention, the medical deviceis used for the delivery of a drug, wherein the drug is the compoundprovided herein, i.e. the compound of formula (I) and in particularleoligin and/or ((di)methoxy)-derivative(s) thereof. Therapeutic systemswhich are used in the art as “drug delivering system”, in particular as“drug eluting stent” contain the agents Paclitaxel (Taxol®) or Sirolimuswhich are structurally not related to the compounds to be used incontext of the present invention. Further, these agents exhibit theireffect in a completely different way compared to the present compoundsof formula (I), and in particular, leoligin and/or its((di)methoxy)-derivative(s).

Paclitaxel promotes the assembly of microtubules from tubulin dimers andstabilizes microtubules by preventing depolymerization. This stabilityresults in the inhibition of the normal dynamic reorganization of themicrotubule network that is essential for vital interphase and mitoticcellular functions Premarket Approval Applications (PMA) of the FDA;P030025: TAXUS™ Express 2™ Paclitaxel-Eluting Coronary Stent System(Monorail and Over-the-Wire). Issued Mar. 4, 2004; Part 2—Summary ofSafety and Effectiveness Data.http://www.fda.gov/cdrh/pdf3/P030025.html. The mechanism (or mechanisms)by which a CYPHER™ Stent (Sirolimus is the active agent containedtherein) exerts its effect on neointima production as seen in clinicalstudies has not been established. Sirolimus inhibits T-lymphocyteactivation and smooth muscle and endothelial cell proliferation inresponse to cytokine and growth factor stimulation. In cells, sirolimusbinds to the immunophilin, FK Binding Protein-12 (FKBP-12). Thesirolimus-FKBP-12 complex binds to and inhibits the activation of themammalian Target of Rapamycin (mTOR), leading to inhibition of cellcycle progression from the GI to the S phase Premarket ApprovalApplications (PMA) of the FDA; P020026: Cypher sirolimus-elutingcoronary stent on the raptor over-the-wire delivery system or raptorrailrapid exchange deliver. Issued Apr. 24, 2003; Part 2—Summary of Safetyand Effectiveness Data. http://www.fda.gov/cdrh/pdf2/P0020026.html.

The compound provided herein, such as compounds of formula (I) and inparticular leoligin, may also be used in liquids intended for risingand/or storing (a) venous bypass(es), particularly before and/or duringbypass operations. The rinsing and/or storage of the venous bypass(es)in such a liquid is particularly advantageous since such a kind of“pretreatment” of the bypass(es) may prevent damage of endothelial cellsand/or inhibit the pathological proliferation of smooth muscle cells,and consequently reduce the probability of hyperplasticdisease/disorder, in particular intimal hyperplasia, stenosis,restenosis or vein-graft disease.

Accordingly, a rinsing and/or storage solution for a venous bypass isprovided herein which comprises the compound of formula (I) and inparticular leoligin and/or its ((di)methoxy)-derivative(s). The storagesolution may, in addition to the compound described herein, comprisefurther components, such as stabilizers, preserving agents, bufferingagents, salts (like NaCl), osmotically active compounds, proteins (likealbumin). It is to be understood that the rinsing and/or storagesolution may comprise only one compound of formula (I) or, optionally,different compounds of formula (I), like leoligin and/or different (a)((di)methoxy)-derivative(s) of leoligin. The rinsing/storage solutionmay comprise the same molar concentration of these different compounds,or, alternatively, different concentrations. For example a firstcompound may be present at a double concentration compared to a secondcompound. Preferably, the molar concentration of the compound in therinsing/storage solution is between 1 and 500 μM, preferably between 10and 200 μM preferably 50 μM. It is envisaged herein that the leaktightness of the venous bypass(es) rinsed with or stored in the solutionas described herein above is to be tested with a device suitable forsuch a test. Leak tightness tests of venous bypass(es) are well known inthe art and a person skilled in the art is therefore aware ofcorresponding means and methods to perform such a test.

The compound(s) of formula (I) may also be applied periadventially to(a) venous bypass(es) before, during and/or after a bypass operation,whereby the compound(s) may, optionally, be applied with a gel (e.g.Pluronic gel) or without a gel.

The present invention also relates to a method for rinsing and/orstoring a venous bypass comprising contacting the venous bypass with thecompound of formula (I), in particular leoligin and/or (a)((di)methoxy)-derivative(s) thereof.

The present invention is further described by reference to the followingnon-limiting figures and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The Figures show:

FIG. 1. Leoligin is a Constituent of Edelweiss (Leontopodium alpinumCass.) Roots

Edelweiss is one of the most popular alpine plants and is also used infolk medicine for the treatment of indigestion, fever, and “abdominalaches”. FIG. 1A shows the flower of Edelweiss (Leontopodium alpinumCass.). FIG. 1B shows the chemical structure of leoligin-IUPAC name:[(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran-3-yl]methyl(2Z)-2-methylbut-2-enoat. Leoligin is a lignan, which was isolated fromthe roots of Edelweiss (Leontopodium alpinum Cass.).

FIG. 2. Leoligin Inhibits Intimal Hyperplasia of Human Saphenous VeinsIn Vitro

The diagram in FIG. 2A summarises the data from experiments where humansaphenous veins were induced to develop intimal hyperplasia in organculture. Tissue samples were incubated with DMSO (solvent control) orvarious concentrations of leoligin for 2 weeks. After the incubationtissues were fixed, dehydrated, and embedded in paraffin. Afterpreparation of sections, samples were stained (Elastica van Giessonstain), and the intimal thickness was measured using Image J software.Approximately 30 measurements per sample and a total of 5 samples(different donors) per concentration were analysed. Shown are meanvalues (in pixel)+/−S.D. Baseline samples were fixed directly afterpreparation of fresh tissue and represent the status of the vessel priorto organ culture.

** . . . p<0.01; *** . . . p<0.001.

FIG. 2B shows the effect of the presence or absence of leoligin on organculture-caused intimal thickening of a representative sample of asaphenous vein with mild pre-existing intimal hyperplasia (upper row,left: baseline; upper row, right, control; lower row, left: leoligin 5μM; lower row, right: leoligin, 50 μM). The area between the arrowsindicates the intimal thickness.

FIG. 3. Leoligin Inhibits SMC Proliferation, Induces a Cell Cycle Arrestin the G1 Phase and Leads to an Accumulation of p27/KIP

In order to investigate the effect of leoligin on the cellular level,isolated primary human vascular smooth muscle cells (SMCs) wereincubated with the indicated concentrations of leoligin for theindicated times. The diagram in FIG. 3A shows an analysis of cellviability determined by the annexin V/propidium iodide method and FACSanalyses. Values shown are mean values from a representative experimentperformed in triplicates. The diagram in FIG. 3B shows an analysis ofcell proliferation by the XTT assay. Values shown are mean values fromthree independent experiments+/−S.D. The histogram blots in FIG. 3C showthe results from a DNA content analysis of control-treated orleoligin-treated SMCs after 24 hours of incubation (upper row, lefthistogram . . . solvent control; upper row, right histogram . . .leoligin). The Western blot (lower row) shows an analysis of p27/KIP ofSMCs incubated with leoligin for 24 hours with the indicatedconcentrations of leoligin.

FIG. 4. Leoligin is not Toxic for ECs and Inhibits TNFalpha-MediatedVCAM Expression.

The effects of leoligin on endothelial cells is shown in FIG. 4. Primaryhuman vascular endothelial cells (ECs) were incubated with the indicatedconcentrations of leoligin for the indicated times. The diagram in FIG.4A shows a representative analysis of cell viability determined by theannexin V/propidium iodide method and FACS analyses. Values shown aremean values from a representative experiment performed in triplicates.The diagram in FIG. 4B shows an analysis of EC proliferation by the XTTassay. Values shown are mean values from three independentexperiments+/−S.D. The impact of leoligin on TNFalpha-induced surfaceexpression of VCAM-1, ICAM-1, and E-selectin (E-Sel) is shown in thethree diagrams in FIG. 4C. Data shown are mean fluorescence intensities(MFI) of representative experiments. The lower right image in FIG. 4Cshows a metabolic protein labelling analysis of EC in the presence ofthe indicated concentrations of leoligin. After protein preparation,total cellular proteins were separated on polyacrylamide gels, gel weredried and analysed by exposure to x-ray films.

FIG. 5. Leoligin Inhibits Neointima Formation In Vivo, without CausingEndothelial Damage.

The effects of leoligin on intimal hyperplasia of venous bypass conduitsin vivo are shown in FIG. 5. The diagram in FIG. 5A displays amorphometric analysis of intimal thickness of vena cava interposed intothe carotid artery of control-treated animals and leoligin-treatedanimals. Before wound closure after the transplantation, aperadventitial depot of 100 μl of 0.9% NaCl (control) or 100 μl of 100μM leoligin in 0.9% NaCl were applied. After 4 weeks conduits wereremoved and subjected to morphometric analyses and toimmunohistochemistry. FIG. 5B: the upper two images show a Elastica vanGiesson stain of sections of the venous conduits. White arrows indicateneointimal thickness which was analysed by morphometry (see FIG. 5A).Central images display a staining for the endothelial cell CD31/PECAM-1antigen (brown, black arrows) combined with a hematoxilin stain. Lowerimages display a immunohistochemical staining of the sections for thecell cycle inhibitor p27/KIP-1 (brown) combined with a hematoxilinstain. The abbreviations “Lu” and “Li” stand for the full terms “lumen”and “liver”, respectively.

FIG. 6. SMC Comparison

The diagram in FIG. 6 shows a comparison of the smooth muscle cell (SMC)proliferation-inhibitory activity of lariciresinol and leoligin by theXTT assay. Values shown are mean values from three independentexperiments+/−S.D.

FIG. 7. SMC Comparison

The diagram in FIG. 7 shows a comparison of the smooth muscle cell (SMC)proliferation-inhibitory activity of leoligin and the two indicatedderivatives by the XTT assay. Values shown are mean values from threeindependent experiments+/−S.D.

FIG. 8. Pathway

FIG. 8 shows a possible synthetic pathway of leoligin.

The following Examples illustrate the invention.

EXAMPLE 1 Leoligin Inhibits Intimal Hyperplasia of Venous Bypass GraftsMaterial and Methods General

All reagents used were of purissimum or analytical grade quality andwere purchased from Sigma Aldrich (Sigma-Aldrich, Vienna, Austria) ifnot specified otherwise. Water was produced by reverse osmosis followedby distillation.

Plant Material, Isolation, and Purification of Leoligin

Ground roots (1907.84 g) from L. alpinum Cass. were exhaustivelymacerated with dichloromethane (12.5 1 DCM, at RT, eight times). Voucherspecimens are deposited at the herbarium of the Institut fërPharmazie/Pharmakognosie, Leopold-Franzens-Universität Innsbruck.Extracts were evaporated to dryness yielding 43.0 g crudedichloromethane extract. 40.0 g of the obtained crude extract wereredissolved in 100 ml MeOH and separated in a MeOH soluble and insolublepart. The soluble part was separated by Sephadex® LH 20 (PharmaciaBiotech, Sweden) CC (90×3.5 cm) with MeOH as mobile phase yielding 8fractions. Fraction 5 (15.13 g; 320-410 ml elution volume) wasrechromatographed by silica CC (180 g, 41×3.5 cm) using a PE-acetonegradient with an increasing amount of acetone yielding 40 fractions (A-1to A-40). A small amount (28.2 mg) of Fraction A-21 (PE/acetone, 85:15;441.2 mg) was separated by semi preparative HPLC (Phenomenex SynergyMax-RP column (10 μm, 10×250 mm); 55% acetonitrile/45% water, isocratic;flow: 3.50 ml/min; 25° C.) yielding 16.0 mg pure leoligin and i4.0 mg ofits 5-methoxy derivative. A small amount (26.5 mg) of Fraction A-22(PE/acetone, 85:15; 77.8 mg) was separated by semi preparative HPLC(Waters X-Terra Prep MS C18, 5 μm, 7.8×100 mm column; 70% MeOH/30%water, isocratic; flow: 1.50 ml/min; 25° C.) yielding 6.3 mg of the5,5′-dimethoxyderivative of leoligin.

Preparation of Extracts Enriched in Leoligin and its Methoxy-Derivative

In order to quantify the content of leoligin and its methoxy-derivativein different extract preparations several extraction procedures wereused. Therefore ground roots (20.00 g) from L. alpinum Cass. wereexhaustively macerated with dichloromethane (100 ml DCM, at RT, eighttimes). After filtration the obtained extracts were combined, evaporatedto dryness to yield a semi solid DCM-extract. Other extracts wereprepared by ultrasonic extraction using 20.00 g ground roots which weresonicated for 15 min using 1×200 ml and 1×100 ml of solvent or a secondultrasonic extraction cycle after air drying of the plant material (5.00g; 2×15 min; 2×100 ml solvent). The leoligin content was determined bymeans of HPLC-quantification using the method of external standard. Eachextract was prepared in duplicate and quantified in triplicate. Thecontent of 5-Methoxy-leoligin was calculated using the calibration curveof leoligin.

Cell Culture

Human umbilical vein endothelial cells (HUVECs) were isolated fromumbilical cords (kindly donated by the Gynecology and ObstetricsDepartment, Innsbruck Medical University) by enzymatic detachment usingcollagenase, as previously described; see Bernhard, FASEB J 17(15),2302-4 (2003). Human umbilical vein smooth muscle cells (SMCs) wereisolated from the same umbilical cords according to Chamley-Campbell,Physiol Rev 59(1), 1-61 (1979). SMCs were routinely passaged in 0.2%gelatine-coated (Sigma, Steinheim, Germany) polystyrene culture flasks(Becton Dickinson, Meylan Cedex, France) in Medium 231 (CascadeBiologics, Paisley, UK). The isolation and analysis of human umbilicalcord ECs and SMCs has been approved by the Ethics Committee of theInnsbruck Medical University.

Quantification of Cell Death and Cellular DNA Content

For detection and/or quantification of cell death, forward/sidewardlight scattering analysis, the AnnexinV-propidium iodide method, andstaining of nuclear DNA content (cell cycle analyses) were used asdescribed; see Bernhard FASEB J 13(14), 1991-2001 (1999).

Analysis of Cellular Proliferation

Cell proliferation was measured by the XTT cell proliferation assay(Biomol, Hamburg, Germany) as described by the manufacturer. The XTTassay is based on the ability of metabolic active cells to reduce thetetrazolium salt XTT to orange coloured compounds of formazan. The dyeformed is water soluble and the dye intensity can be read at a givenwavelength with a spectrophotometer. The intensity of the dye isproportional to the number of metabolic active cells. In addition to theXTT assay all proliferation-experiments were also evaluated by countingcells in a coulter counter.

Surface Expression Analyses ICAM, VCAM, and E-Selectin.

FACS-based analyses of surface adhesion molecule expression wasperformed according to a protocol by Gräbner et al. (see Grabner,Cytometry 40(3), 238-44 (2000). Antibodies used were anti-VCAM-1antibody (clone 1.4 C3, Neomarkers), anti-ICAM-1 antibody (clone 28,DAKO Cytomation), and anti-E-selctine antibody (clone 16G4; Novocastra).

Western Blotting

Western blotting was performed as previously described; see Bernhard,Cell Death Differ 8(10), 1014-21 (2001). Primary antibodies used wereanti-p27/KIP-1 antibody (clone 57; BD Transduction Laboratories).

Metabolic Labelling of Proteins

Metabolic labelling with ³⁵S-methionine/cysteine was performed aspreviously described (Bernhard (2001), loc. cit.).

Human Saphenous Vein Organ Culture

For organ culture experiments, remnants (surgical waste) of saphenousveins from patients undergoing CABG were collected. The saphenous veinswere opened longitudinally and attached to silicone patches (with theendothelium facing upwards). Tissue pieces were incubated with culturemedium (RPMI1640, 30% serum, 8 heparin mU/ml, antibiotics) for 2 weeksin the presence or absence of leoligin. to induce intimal hyperplasia.For details see Schachner, Eur J Cardiothorac Surg 32, 906-911 (2007).Leoligin was added freshly every second day to organ cultures over atime period of 2 weeks. 5 μM leoligin completely inhibited intimalhyperplasia (p=0.003), and 50 μM even reversed pre-existing intimalhyperplasia of saphenous veins (p<0.001). The use of human saphenousveins has been approved by the Ethics Committee of the Innsbruck MedicalUniversity.

Mouse Model—Vein Graft Disease

In the applied mouse model the vena cava of a donor mouse was interposedinto the carotid artery of a recipient mouse. After transplantation andprior to wound closure a 100 μl depot of 0.9% NaCl (control group) or100 μM leoligin in 0.9% NaCl (leoligin group) was placed around theadventitia of the vein graft in the recipient mouse. 4 weeks after theintervention mice were sacrificed and the interposed venae cavae wereharvested for analyses. Of the 7 animals per group 3 control and 2leoligin-treated animals showed no blood flow due to thromboses after 4weeks and were consequently excluded from further analyses. For detailson the surgical procedure see Schachner, Eur J Cardiothorac Surg 30(3),451-63 (2006); Schachner, Heart Surg Forum 9(1), E515-E517 (2006).Animal experiments were approved by the Commission for Animal TestingAffairs of the Austrian Ministry for Science and Research.

IHC and Morphometric Analyses

Following fixation in 4% paraformaldehyde and dehydration of tissuesfrom organ culture and mouse experimentation, tissues were embedded inparaffin (venae cavae from the in vivo experiments were embedded intomouse liver prior to fixation) and sections were prepared.Immunohistochemistry was performed with the Accustain Elastica Stain(HT25) kit (Sigma-Aldrich, USA) or the En Vision+System-HRP (DAB)(DakoCytomation, Denmark) according to the manufacturers instructions.Primary antibodies used were anti-p27/KIP-1 antibody (clone 57; BDTransduction Laboratories), and anti-CD31/PECAM-1 antibody (clone JC70A,Dako Cytomation). Image analyses were performed using the Image Jsoftware of the National Institute of Health (USA).

Results Preparation of Extracts Enriched in Leoligin and IstMethoxy-Derivates

Root extracts comprising leoligin and its derivates 5-Methoxy-leoliginund 5,5′-dimethoxy-leoligin have been prepared as described herein above(i.e. roots macerated at room temperature and extracted usingdichlormethane), whereby the yield of the extract lies typically in therange of between 1.03 bis 2.26% and whereby the maximum level of theleoligin and methoxy-leoligin content (quantified as a mixture thereof)is 2.14%.

In order to obtain extracts enriched in leoligin and its derivates, theplant material is in a first step extracted with hexane or heptanefollowed by a subsequent extraction with organic solventsdichloromethane, chloroform or ternary butyl methyl ether. The resultsare summarized in the table below.

Total lignan Pre- Leoligin content in treatment Yield of content theextract solvent Extraction extract (w %) in (calculated as (defatting)solvent (w %) the extract leoligin; w %) — dichloro- 1.03-2.26%0.77-1.36% 1.69-2.14% methane* — n-hexane** 0.07-0.15% 0.67% 1.47% —n-heptane** 0.12-0.27% 0.74% 1.55% n-hexane dichloro- 0.50% 1.32% 2.67%methane*** n-hexane chloroform*** 0.65% 1.31% 2.71% n-hexane tBMe***0.39% 1.19% 2.43% *exhaustive mazeration (20.00 g; 8 × 100 ml);**ultrasonic extraction (20.00 g; 15 min; 1 × 200 ml; 1 × 100 ml);***ultrasonic extraction (5.00 g; 2 × 15 min; 2 × 100 ml).

“Yield of extract” refers to the weight of the extract vs. the basicmaterial used, where a leoligin content refers to the percentage byweight of Leoligin and lignan, respectively, in the extract.

The concentration of lignans (and derivatives) was also increased by theuse of Sephadex-LH20-column chromatography (increase in the leoligincontent from 0.77% to 2.21%). The most pronounced increase (increase inthe leoligin content from 1.36% auf 9.76%) was achieved using silica gelcolumn chromatograph (mobile phase: Petroleum ether-aceton).

Leoligin a Compound from Edelweiss Potently Inhibits Intimal Hyperplasiain a Human Saphenous Vein In Vitro Model.

Leoligin[(2S,3R,4R)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran-3-yl]methyl(2Z)-2-methylbut-2-enoat (see FIG. 1) a compound that was previouslyisolated from the roots of Edelweiss (Leontopodium alpinum Cass.) is alignan type secondary plant metabolite. In our organ culture-basedscreen for compounds capable of inhibiting human saphenous vein intimalhyperplasia, leoligin showed a profound inhibitory activity (see FIG.2). Leoligin when added to organ cultures over a time period of 2 weeks(added freshly every second day) inhibited intimal hyperplasia in adose-dependent fashion. 5 μM leoligin completely inhibited intimalhyperplasia (p=0.003), and 50 μM even reversed pre-existing intimalhyperplasia of saphenous veins (p<0.001) (see FIG. 2).

Leoligin Inhibits SMC Proliferation by Inducing a Cell Cycle Arrest inthe G1 Phase which is Associated by a Shift in Molecular Weight and anAccumulation of p27/KIP Protein

In order to reveal the mechanism underlying leoligin-mediated inhibitionand reversal of saphenous vein intimal hyperplasia we analysed theeffects of leoligin on isolated primary human vascular smooth musclecells, which represent the central cell type in intimal thickening andintimal hyperplasia (SMC proliferation, and migration). Our analysesclearly showed that leoligin causes only a marginal increase in celldeath (apoptosis and necrosis) in SMCs after 48 hour (FIG. 3, upperpanel, left side). Analyses at later time points were consistent withthe findings after 48 hours (data not shown). In contrast, analyses ofcell numbers by the XTT proliferation assay (FIG. 3, upper panel, rightside) as well as by Casy-based cell counting (data not shown) revealed asignificant SMC proliferation inhibitory effect of leoligin. Consequentmeasurements of nuclear DNA content (FIG. 3, two lower panels, leftside) revealed that leoligin causes a massive accumulation of cells inthe G1-phase of the cell cycle. Since it is well known that a number ofcell cycle regulators can cause a G1 arrest, we performed a Western blotbased analyses for changes in G1 arrest-relevant cell cycle regulators.The lower right panel of FIG. 3 shows a Western blot demonstrating thatleoligin induces a complete change in the appearance of p27/KIP proteini.e. from one signal at 27 kD to an intensive signal at 58 kD, and threeweak signals at 24, 27 (original signal) and 85 kD (the 85 kD signal washardly visible and is not shown). The accumulation of p27/KIP is wellknown to mediate a G1-arrest by binding to and thereby inactivating thecyclin D1/E-cdk2 complex. The observed shift in molecular weight mayindicate the binding of p27/KIP to this complex or an oligomerisation ofp27/KIP molecules.

Leoligin is not Toxic for ECs and Inhibits TNFalpha-Mediated VCAM-1Expression.

Since the integrity of the vascular endothelium plays a central role invascular repair and healing, anti-thrombosis, and the graftatherosclerosis-relevant control of cell (macrophages) and compound(cholesterol) exchange between the blood and the vessel wall we alsoanalysed the effects of leoligin on the vascular endothelium. Althoughleoligin also inhibited the proliferation of ECs no toxic or celldeath-inducing effect of leoligin on ECs could be observed (see FIG. 4,left panels). Analyses at later time points were consistent with thefindings after 48 hours (data not shown). Interestingly, leoligininhibited TNFalpha-mediated surface expression of VCAM-1 (upper rightpanel). To exclude that general phenomena, like an inhibition of theendothelial translational machinery by leoligin accounts for thisobservation, we performed metabolic protein labelling experiments (FIG.4, lower right panel), and analysed the effect of leoligin on otheradhesion molecules (FIG. 4, right side, two central panels). These datademonstrate that leoligin does not block protein synthesis, and does notinterfere with the translocation of proteins to the cellular surface ingeneral, but specifically inhibits stimulated VCAM-1 expression on theEC surface.

Leoligin Inhibits Neointima Formation In Vivo, without CausingEndothelial Damage.

To test the applicability of leoligin in vivo we analysed leoligineffects in a mouse model for vein graft disease (see Methods section).Like in human saphenous vein bypass conduits the transplant develops asevere intimal hyperplasia within a couple of weeks. Leoligin wasapplied directly after the transplantation as a periadvential reservoir(100 μl of 100 μM leoligin in 0.9% NaCl) before surgical closure of thewound. Four weeks after the transplantation mice were sacrificed and thevenous conduits were removed for further analyses. Thrombosed veins wereexcluded form further analyses (see Methods section) and the remainingsamples were analysed by means of immunohistochemistry. FIG. 5 (leftside) shows that leoligin treatment, in contrast to control-treatment(0.9% NaCl), potently inhibited intimal hyperplasia in vivo. Vein graftsof mice treated with leoligin showed significantly reduced neointimalthickness (upper central and upper right image). A CD31 (endothelialmarker) staining of the sections revealed that the endothelial layer ofleoligin and control treated conduits was intact. Finally, a p27/KIPstaining of the sections revealed that a large number of cells/nuclei inthe leoligin-treated grafts stained positive for p27/KIP even four weeksafter the application of leoligin, indicating a long duration of drugeffect.

In the in vivo experiments described herein above leoligin was addeddirectly after the surgical procedure. Thereafter, mice were kept foradditional 4 week, only then samples were analysed. Since a large numberof SMCs in the leoligin group stained positive for p27/KIP the mechanismvia which leoligin inhibits the proliferation of SMCs may be theinduction of differentiation. In contrast to cell culture and in vivoexperiments where leoligin was added only once and at the beginning ofthe experiments, leoligin showed profound inhibition of intimalhyperplasia in the in vitro organ culture experiments (leoligin wasadded every second day) already at a concentration of 5 μM. These datasuggest that a constant addition of leoligin may improve the results butalso that a positive therapeutic effect can be achieved by a singleapplication of leoligin, the consequence of which being detectable even4 weeks after this application in vivo. With respect to the used ofsaphenous veins with pre-existent intimal hyperplasia for CABG, leoliginmay help to increase patency rates simply by applying leoligin to thegrafts prior to re-implantation. In addition, differences in thepotential to inhibit or reverse intimal hyperplasia by varying the doseand duration of treatment, may help to reduce complications e.g. graftaneurysms which may occur as a result of reduced adaptive tissueremodelling in the vessel wall.

The above provided assessment using an in vivo mouse model for veingraft disease can also be carried out in larger animals/animal models.An exemplary protocol (experimental set up and evaluation of data) usinga porcine animal model (i.e. “Landschwein”) to assess the efficacy ofthe particular compound known under the trivial name “Leoligin” is givenherein below.

Experimental Setup

Leoligin is used in a concentration of 100 μM. Animals are separatedinto two treatment groups (control, treatment). Furthermore, animals areseparated into groups for a harvesting time point after 4 weeks and 12weeks, respectively. The treatment schedule is illustrated in thefollowing table:

Time of analysis Number Compound tested (postoperativ) of animalsTreatment group 1 Control NaCl  4 weeks 6 Leoligin NaCl + Leoligin  4weeks 6 Total 12 Treatment group 2 Control NaCl 12 weeks 6 LeoliginNaCl + Leoligin 12 weeks 6 Total 12

Anaesthesia and Surgical Procedure—Vena Saphena as Carotis Graft in the“Landschwein” (Pig Race)

For transportation and to prevent narcosis complications animals receivein the cot an intramuscular injection of 4 mg/kg Azaperon and 0.1 mg/kgAtropin one hour prior to narcosis. Sleeping animals are transported ina transport box with straw filler to the operating theatre. Animalsreceive an infusion of 2-3 mg/kg Propofol and 15 mg Piritramid via theear vein, followed by intubation and ventilation with 30% O₂. Animalsreceive an initial muscle relaxation 8 mg Pancuronium and repeatedly 0.2mg/kg/h Pancuronium. For maintenance of narcosis animals receive apermanent infusion of 8-12 mg/kg/h Propofol und 15 mg Piritramid. Inorder to prevent pain in the animals the dose may be increased. Duringthe preparative phase animals receive in addition to the above apermanent infusion with 6 ml/kg/h of Ringer-Lactate.

Surgical Procedure: Carotis-Graft

After the initiation of anaesthesia according to the above protocolanimals are kept in dorsal position, shaved, washed with Octinsept fordisinfection, and covered with sterile cloth.

For preparation of the saphenous vein a ca. 10 cm longitudinal incisionis made on the hind leg of animals. Following the identification of thevein, the vein is cleaned from surrounding tissue by“no-touch”-technique. Side arms are clipped and cut through. After thepreparation of the vein by the above technique, the vein is removed andcannulated via the distal end. To test for tightness and to expand thevessel diameter to the proper size, the vein is expanded by flushingwith a pressure of 80-100 mmHg. Flushing and expansion of the sephenousvein in control animals is done using 0.9% NaCl-solution including 1‰DMSO, in the treatment group a 100 μM leoligin (or derivative thereof)solution in 0.9% NaCl-solution including 1‰ DMSO is used(leoligin/derivative is first solubilised in DMSO (100 mM solution) andis then diluted 1:1000 in 0.9% NaCl-solution.). After expansion, veinsare incubated for 30 minutes to one hour at room temperature in theabove solutions. After hind leg wound closure, the surgeon starts withthe preparation of the neck region. First an incision is placed on theright jugular side medial of the musculus stemocleidomastoideus. After ablunt preparation and cut through of the platysma the trionum caroticumis identified. Under protection of the vena jugularis interna and nervusvagus, the arteria carotis communis is prepared, and a 3 cm segment isisolated. Then 100 units of heparin/kg are injected intravenously. Afterplacing of a vessel clamp at the proximal and distal side of thesegment, the arteria carotis communis is cut through and the surgicalmargin is trimmed to an angle of 45°. A part of the prepared isolatedvena saphena magna is shortened to the corresponding length and isanastomosed on both sides by an end-end technique. The anastomose issutured with 7/0 Prolene in continuos technique. To increase theexposure time of the vessel to leoligin (or derivative), resorbablehaemostypticum (Dabostemp clothes) are soaked with 0.9% NaCl-solutionincluding 1‰ DMSO (control), or a 100 μM leoligin (or derivative)solution in 0.9% NaCl-solution including 1‰ DMSO (treatment group) arewarped around the interposed vein. The wound is closed in layers, thenthe skin is closed.

To keep the number of animals low the same procedure is repeated on theleft side according to the above protocol. After surgery, animals aretransported to the animal keeping facility in a species appropriatemanner and receive a pain therapy for additional 7 days with Dipidolorand Novalgin.

Harvesting-Surgery

Harvesting is performed 4 and 12 weeks after surgery. Anaesthesia andtransport, washing and covering with sterile clothes is accomplished asdescribed above. First the surgical scar on the right side is locatedand the site is re-opened. After the preparation of the interposed veinvessel claps are places on the proximal and distal site, the vein isremoved. The same procedure is accomplished on the left side. Animalsare euthanized with a bolus of Dormicum and Fenta, followed by aninjection of 20 mVal KCl.

Analyses

The following analyses are performed on the harvested vein graft:

-   -   Histology: VanGiessen, Oil red, HE-staining;        Immunohistochemistry: β-Aktin, p27, p21, CD31, ICAM-1, VCAM-1,        CD3, CD4, CD8, CD56, etc. Analysis is performed using the Image        J Software. Evaluation and data analyses will be performed by        blinded researchers; see Reisinger (2009) Cardiovasc Res. 82;        542-549, and Messner (2009), Arterioscler. Thromb. Vasc. Biol.    -   Electron microscopical analyses (SEM/TEM) of the endothelial        surface and the composition of the vessel wall; see Messner        (2009), Arterioscler. Thromb. Vasc. Biol., and Bernhard (2003),        FASEB J. 17:2302-2304.    -   Contractility and functionality of grafts are analysed in an        organ bath; see Hammerer-Lercher A, Clin Sci (Lond). 2006;        111:225-231. Prior to harvesting the intima media thickness of        grafts is analysed by sonography; see Knoflach (2003)        Circulation; 108:1064-1069, and Knoflach (2009) Stroke;        40:1063-1069.    -   Collection of blood and serum samples for the detection of the        test compound (i.e. leoligin).

Based on the above analyses, the following parameters/effects defineindependently of each other a treatment success by Leoligin (orderivative): 1) An intima thickness and/or intima-media thickness of thetreatment group below the control group. 2) A smaller number of smoothmuscle cells in the intima of the treatment group compared to thecontrol. 3) The presence of a higher number of p27 and or p21 positivecells in the treatment group compared to the control. 4) A smallerdegree of neointima formation in the treatment group compared to thecontrol. 5) A reduced presence of tissue remodelling processes in thetreatment group compared to the control. 6) A lower number ofpro-inflammatory cells in the vessel wall in the treatment groupcompared to the control. 7) An intact endothelium. 8) A physiologicaldegree of contractility of the vessels. 9) A low degree of adhesionmolecule expression on the endothelial surface. 10) patency of graftsand no signs of thrombus formation. 10) Conserved elasticity of graftsin the treatment group compared to the control. And 11) Conservedcontractility of grafts in the treatment group compared to the control.

EXAMPLE 2 Leoligin is a Stronger Inhibitor of SMC (Smooth Muscle CellProliferation) than Lariciresinol

The diagram in FIG. 6 shows a comparison of the smooth muscle cell (SMC)proliferation-inhibitory activity of lariciresinol and leoligin by theXTT assay after 72 h. Values shown are mean values from threeindependent experiments+/−S.D. The obtained IC₅₀ value of leoligin (54.5μM; CI₉₅: 49.4-59.4 μM) was found to be more effective than thelariciresinol (IC₅₀>100 μM).

Methods FIG. 6 Analysis of Cellular Proliferation

As described above (Example 1) cell proliferation was measured by theXTT cell proliferation assay (Biomol, Hamburg, Germany) as described bythe manufacturer. The XTT assay is based on the ability of metabolicactive cells to reduce the tetrazolium salt XTT to orange colouredcompounds of formazan. The dye formed is water soluble and the dyeintensity can be read at a given wavelength with a spectrophotometer.The intensity of the dye is proportional to the number of metabolicactive cells.

A use of lariciresinol in the context of stent implantation has alsobeen proposed in DE 10 2004 046 244.

EXAMPLE 3 Leoligin and its Methoxy-Derivatives Inhibit SMC Proliferation

The diagram in FIG. 7 shows a comparison of the smooth muscle cell (SMC)proliferation-inhibitory activity of leoligin and its naturalderivatives by the XTT assay after 72 h. Values shown are mean valuesfrom three independent experiments+/−S.D. The obtained IC₅₀ value ofleoligin (54.5 μM; CI₉₅: 49.4-59.4 μM) was found to be not significantdifferent from those of 5′methoxyleoligin (45.9 μM; CI₉₅: 37.3-53.9 μM)or 5,5-dimethoxyleoligin (IC₅₀ 48.6 μM; CI₉₅: 39.9-56.6 μM).

Methods FIG. 7 Analysis of Cellular Proliferation

As described above (Example 1) cell proliferation was measured by theXTT cell proliferation assay (Biomol, Hamburg, Germany) as described bythe manufacturer. The XTT assay is based on the ability of metabolicactive cells to reduce the tetrazolium salt XTT to orange colouredcompounds of formazan. The dye formed is water soluble and the dyeintensity can be read at a given wavelength with a spectrophotometer.The intensity of the dye is proportional to the number of metabolicactive cells.

1. (canceled)
 2. The method of claim 9, wherein the compound of formula(I) has the stereochemistry indicated in formula (Ia):

wherein R¹ to R⁷ and X are defined as in claim
 9. 3. The method of claim9, wherein R⁷ is —OC(O)R⁹ or —C(O)OR⁹.
 4. The method of claim 9, whereinat least one of R¹, R² and R³ and at least one of R⁴, R⁵ and R⁶ is analkoxy group.
 5. The method of claim 9, wherein at least two of R¹, R²and R³ and at least two of R⁴, R⁵ and R⁶ are alkoxy groups.
 6. Themethod of claim 9, wherein the compound of formula (I) has the followingstructure:


7. The method of claim 9, wherein five or all six of R¹ to R⁶ arealkoxy, and the remaining group of R¹ to R⁶, if applicable, is hydrogen.8. (canceled)
 9. A method for treating or ameliorating a hyperplasticdisease/disorder comprising the administration of a compound of formula(I) to a subject in an amount effective to treat or ameliorate suchdisease/disorder:

wherein: R¹, R² and R³ are independently selected from H, OH, halogen,alkyl, or alkoxy; and R⁴, R⁵ and R⁶ are independently selected from H,OH, halogen, alkyl, or alkoxy; R⁷ is selected from —OR⁸, —N(R^(8′))R⁸,—SR⁸, —C(O)R⁸, —OC(O)R⁹, —C(O)OR⁹, —N(R^(9′))C(O)R⁹, —C(O)N(R^(9′))R⁹ or—S(O)R⁹; wherein R⁸ and R⁹ are independently selected from alkyl oralkenyl and R^(8′) and R^(9′) are independently selected from H, alkylor alkenyl; and wherein any alkyl or alkenyl group comprised in R⁷ maybe unsubstituted or substituted by one or more substituents, selectedfrom OH, halogen or alkoxy; and X is selected from O, S, C(R¹⁰)R¹⁰ andNR¹⁰, wherein R¹⁰, independently for each occurrence, is H, alkyl oralkenyl; and the dashed lines in the ring structure containing the groupX indicate that the respective bond may be a single or a double bond; orany pharmaceutically acceptable salt or solvate thereof.
 10. The methodof claim 9, wherein said hyperplastic disease/disorder is hyperplasia.11. The method of claim 10, wherein said hyperplasia is intimalhyperplasia.
 12. The method of claim 11, wherein said intimalhyperplasia is stenosis or restenosis.
 13. The method of claim 11,wherein said intimal hyperplasia is atherosclerosis.
 14. The method ofclaim 9, wherein said hyperplastic disease is a proliferative orneoplastic disease.
 15. The method of claim 14, wherein saidproliferative disease is a benign proliferative disease.
 16. The methodof claim 15, wherein said benign proliferative disease is benignproliferative breast disease.
 17. The method of claim 14, wherein saidproliferative disease is a cancerous disorder.
 18. The method of claim17, wherein said cancerous disorder is selected from the groupconsisting of leukemia, myelotic cancer, B-cell lymphomas, prostatecancer, breast cancer, colon cancer, lung cancer and skin cancer.19.-23. (canceled)
 24. The method of claim 9, whereby said compound isadministered by any one of a parenteral route, oral route, intravenousroute, subcutaneous route, intranasal route or transdermal route. 25.The method of claim 9, wherein said subject is a human. 26.-32.(canceled)